NUCLEAR  AND  
CYTOPLASMIC  
STAINING  
 
Presented  by:  
Jacqueline  Brooks  
MB,  HTL,  SCT  (ASCP)  Q  IHC,  CM  IAC  
Cunningham  Pathology  
jbrooks@cunninghampathology.com  
 

1  
Housekeeping  

2  
Cell  Morphology  

3  
Cell  Membrane  
Cell  Membrane    
The  outer  boundary  of  the  cell.  The  cell  
membrane  helps  control  what  substances  
enter  or  exit  the  cell.  Is  composed  mainly  of  
protein  and  lipid  molecules.  

4  
 Endoplasmic  ReXculum  
Endoplasmic  re2culum  
The  Endoplasmic  reXculum  is  a  network  of  membranes  in  eukaryoXc  cells  
which  helps  in  control  of  protein  synthesis  and  cellular  organizaXon.  A  
network  of  interconnected  membranes  forming  sacs  and  canals.  
Transports  materials  within  the  cell,  provides  a\achement  for  ribosomes,  
and  synthesizes  lipids.  

5  
 Golgi  apparatus  

Intracellular stack of membrane bounded vesicles in which glycosylation

and packaging of secreted proteins takes place.

6  
 Ribosomes  
 
ParXcles  composed  of  
protein  and  RNA  molecules.  
FuncXon  to  synthesize  
proteins.  

7  
 Mitochondria  

A small intracellular organelle which is responsible for energy

production and cellular respiration.

8  
 Lysosomes  
A class of morphologically heterogeneous cytoplasmic particles in
animal and plant tissues latency of these enzymes. The intracellular
functions of lysosomes is characterised by their content of hydrolytic
enzymes and the structure-linked is depend on their lytic potential.
The single unit membrane of the lysosome acts as a barrier between
the enzymes enclosed in the lysosome and the external substrate.
The activity of the enzymes contained in lysosomes is limited or nil
unless the vesicle in which they are enclosed is ruptured. Such
rupture is supposed to be under metabolic (hormonal) control.

9  
 Peroxisomes  
LYSOSOMES  AND  peroxisomes,  THE  CELL'S  
"DIGESTIVE  SYSTEM"  When  a  white  blood  cell  
engulfs  a  bacterium  and  destroys  it,  the  white  
cell's  lysosomes  do  most  of  ..."  
 

10  
 Centrosome  
An  organelle  located  in  the  cytoplasm  of  all  animal  cells  and  many  plants,  
fungi,  and  protozoa  that  controls  the  polymerizaXon,  posiXon,  and  polar  
orientaXon  of  many  of  the  cell's  microtubules  throughout  the  cell  cycle.    

11  
Cilia  and  Flagella  
The  flagella  is  a  tail  like  appendage  that  propels  
the  cell.  Both  the  cilia  and  flagella  are  a\ached  
to  the  outside  of  the  cell.    

12  
Microfilaments  and  Microtubules  

The  cytoskeleton  (also  CSK)  is  a  cellular  "scaffolding"  or  "skeleton"  

contained  within  the  cytoplasm  and  is  made  out  of  protein.  The  
cytoskeleton  is  present  in  all  cells;  it  was  once  thought  to  be  unique  to  
eukaryotes,  but  recent  research  has  idenXfied  the  
prokaryoXc  cytoskeleton.  It  has  structures  such  as  flagella,  cilia  and  
lamellipodia  and  plays  important  roles  in  both  intracellular  transport  
(the  movement  of  vesicles  and  organelles,  for  example)  and  
cellular  division.    

13  
 Nuclear  Envelope  
nuclear  envelope  The  double  membrane  that  separates  the  nucleoplasm  
(see  nucleus)  of  a  cell  from  the  cytoplasm.  The  membranes  consist  of  
lipid  bilayers  that  are  separated  by  a  perinuclear  space  (or  compartment).  
The  outer  membrane  is  conXnuous  with  the  rough  endoplasmic  reXculum  
and  is  structurally  and  funcXonally  disXnct  from  the  inner  membrane.  The  
envelope  is  perforated  at  intervals  by  nuclear  pores,  which  provide  a  
channel  for  the  selecXve  transfer  of  water-­‐soluble  molecules  between  the  
nucleus  and  the  cytoplasm.  Each  nuclear  pore  is  surrounded  by  a  disc-­‐
shaped  structure  (nuclear  pore  complex)  consisXng  of  an  octagonal  
arrangement  of  eight  protein  granules.  

14  
 Nucleolus  
 A  small  dense  body  (sub  organelle)  within  the  nucleus  of  eukaryoXc  cells,  
visible  by  phase  contrast  and  interference  microscopy  in  live  cells  
throughout  interphase.  Contains  RNA  and  protein  and  is  the  site  of  
synthesis  of  ribosomal  RNA.  The  nucleolus  surrounds  a  region  of  one  or  
more  chromosomes  (the  nucleolar  organiser)  in  which  are  repeated  
copies  of  the  DNA  coding  for  ribosomal  RNA.    

15  
 ChromaXn  
Stainable  material  of  interphase  nucleus  consisXng  of  nucleic  acid  
and  associated  histone  protein  packed  into  nucleosomes.  
EuchromaXn  is  loosely  packed  and  accessible  to  RNA  polymerases,  
whereas  heterochromaXn  is  highly  condensed  and  probably  
transcripXonally  inacXve.    

16  
 Cancer  

Atypical  adenomatous   Loss  of  structural  differenXaXon  

hyperplasia  of  the  prostate   within  a  cell  or  group  of  cells  omen  
with  increased  capacity  for  
mulXplicaXon,  as  in  a  malignant  
tumor.    

17  
Metastasis  

18  
Cause  or  Causes  of  Cancer  

19  
 H&E  Stain  
1.  FixaXon  
2.  Processing  
3.  Embedding  
4.  Microtomy  
5.  Slide  Drying  
6.  DeparaffinizaXon  
7.  HydraXon  
8.  Staining  (Hematoxylin,  differentaXon,  bluing,  Eosin)  
9.  DehydraXon  
10.  Clearing  
11.  Coverslipping  

20  
Skin    

21  
Skin  

keraXnized    
squamous  
epithelium  

22  
Skin  

Nucleus  open  chromaXn  

23  
STAINING  MECHANISMS  
 

Most  staining  reacXons  involve  both  physical  and  chemical  factors.  The  
fat  stain  is  an  example  of  a  purely  physical  stain,  with  the  dye  absorbed  
(soaked  up)  by,  and  dissolved  in,  the  lipid.    

Chemical  bonding  of  the  dye  to  the  Xssue:  

Ionic  or  electrostaXc  bonding  occurs  when  the  dye  and  the  substance  to  
be  dyed  develop  different  charges  and  thus  become  a\racted  to  each  
other.  

Hydrogen  bonding  occurs  when  covalently  bonded  hydrogen  is  

a\racted  to  atoms  that  have  a  strong  electronegaXve  charge.  

Covalent  bonding  occurs  when  atoms  share  electrons.  

Van  der  Waals  forces  are  caused  by  the  electrostaXc  a\racXon  of  a  
molecule  to  the  electrons  of  its  neighboring  molecules.

24  
 Skin  /  RBC’s  

Blood  vessel   RBC’s  

nucleus  

25  
Prostate  Bx  High  power  

Nucleus  open  chromaXn  

26  
NUCLEAR  STAINING  

(DNA  in  the  nucleus  gives  it  a  net  negaXve  charge)  

Nuclear  staining  is  not  fully  understood,  but  apparently  

occurs  through  2  different  mechanisms.  

• Staining  done  with  the  basic  (caXonic  or  posiXvely  

charged)  dyes  

• Staining  done  with  dyes  combined  with,  or  followed  

by,  metal  mordants  

Basophilic  –  An  acidic  (anionic,  negaXvely  charged)  

substance  that  is  easily  stainable  with  basic  (caXonic,  
posiXvely  charged)  dyes

27  
 Prostate  Bx  low  power  

Open  gland  

Open  gland  

28  
 Cytoplasmic  Staining  
 
•  (proteins  are  in  the  cytoplasm  giving  it  a  net  posiXve  charge)  
•  Acidophilic  –  A  basic  (caXonic,  posiXvely-­‐charged)  substance  that  is  
easily  stainable  with  acid  dyes.  An  example  is  cell  cytoplasm,  which  
is  readily  stainable  with  the  acid  (anionic,  negaXvely  charged)  dye  
eosin.  
•  If  the  eosin  soluXon  is  not  below  pH  6,  the  eosin  will  not  a\ach  to  
the  cytoplasmic  proteins  because  above  pH  6,  the  proteins  will  
have  a  net  negaXve  charge.  
•  IEP  (isoelectric  point)  (the  point  where  the  posiXve  and  negaXve  
charges  are  equal)  The  IEP  of  proteins  is  approximately  pH  6;  
below  the  IEP  or  below  pH  6,  the  net  charge  on  the  nonnuclear  
proteins  is  posiXve  and  the  a\racXon  is  for  an  anionic  dye  above  
the  IEP,  the  net  charge  is  negaXve  and  the  a\racXon  is  for  a  
caXonic  dye.  

29  
Prostate  bx  low  power  
normal  
nuclei  

Stroma  

Open  gland  

30  
 The  Dyes  
 
•  Chromophore-­‐The  chemical  grouping  that  bestows  the  property  of  color  on  
a  compound  

•  Chromagen  –  A  benzene  derivaXve  containing  a  color-­‐bearing  group,  or  

chromophore  

•  Auxochrome  –  The  chemical  group  present  in  a  dye  that  causes  it  to  bind  to  
certain  Xssue  elements.  This  group  can  develop  a  charge  (+  or  -­‐)  and  thus  
bind  to  oppositely-­‐charged  groups  present  in  the  Xssue.  The  amino  (-­‐NH2)  
and  the  carbgoxyl  (-­‐COOH)  groups  are  frequently  occurring  auxochromes.  
   
•  Amphoteric  –  Describes  a  substance  that  is  capable  of  acXng  as  either  a  
base  or  an  acid,  depending  on  the  pH  of  the  soluXon.  
   

31  
 Breast  Xssue  

Stroma  

Fat  cell  nucleus  

Fat  cell    

32  
Poorly  Diff  breast  ductal  
CA  

Stroma  

Crowded  nuclei  

Cells  filling  duct  

33  
 Poorly  Diff  Ductal  CA  /  
breast  
Duct  

Duct  

Irregular  cells  filling  duct  

34  
 Factors  AffecXng  Dye  
Binding  
 
•  pH  
•  temperature  
•  concentraXon  
•  salt  content  
•  fixaXve  (formalin  react  with  the  NH2  group,  
because  this  is  the  primary  group  for  binding  
eosin,  Xssue  fixed  in  formalin  will  bind  less  eosin  
than  when  fixed  in  some  of  the  other  soluXons.  
   

35  
 Gallbladder  

Lumen  

RBC’s  

Glandular  cells    

Smooth  muscle  

36  
 Gallbladder    

RBC’s  

Glandular  Cell  

Lumen  

37  
 Gallbladder  low  power  
Serosal  surface  

Lumen  

Mucosal  surface  

RBC’s  

Smooth  muscle  

38  
 The  Nuclear  Dyes  
 
•  Hematoxylin  –  the  most  widely  used  nuclear  stain,  it  is  extracted  from  logwood  a  tree  indigenous  to  
Central  America.  The  freshly  cut  wood  is  colorless  but  becomes  dark  reddish  brown  when  exposed  to  
atmospheric  oxidaXon;  the  oxidized  dye  is  hematein.  SyntheXc  hematoxylin  is  also  available  but  most  
laboratories  sXll  use  soluXons  prepared  from  the  natural  product.  

•  It  is  very  important  to  remember  that  hematoxylin  is  not  a  dye;  hematein,  the  oxidaXon  product  of  
hematoxylin,  is  a  weak  anionic  dye.  OxidaXon  of  hematoxylin  is  necessary  and  may  be  achieved  naturally  
by  exposing  the  soluXon  to  atmospheric  oxygen,  or  by  using  oxidizing  agents  such  as  sodium  iodate,  
mercuric  oxide,  and  potassium  permanganate;  this  oxidaXon  process  is  also  called  ripening.  SoluXons  
should  always  contain  some  un-­‐oxidized  hematoxylin  because  the  process  of  ripening  conXnues  with  
atmospheric  oxidaXon,  and  complete  oxidaXon  or  over  oxidaXon  lead  to  a  breakdown  of  the  soluXon  and  
the  loss  of  good  staining.  

•  Oxidized  hematoxylin  (hematein)  has  li\le  affinity  for  Xssue  but  becomes  a  strong  dye  with  a  parXcular  
affinity  for  nuclei  when  combined  with  a  metallic  mordant.    

•  The  mordant-­‐dye  combinaXon  is  called  a  lake,  and  the  most  commonly  used  hematoxylin  lakes  are  
combinaXons  of  hematein  with  either  aluminum  or  iron.    
   

39  
 Hematein  vs  HemaXn  
•  Hematein  –  The  oxidaXon  product  of  hematoxlin  
and  the  acXve  staining  ingredient  in  hematoxylin  
soluXons.  Hematein  is  the  acXve  dye  formed  by  
the  acXon  of  either  light  and  air  or  a  chemical  
oxidizing  agent  on  hematoxylin.  
•  HemaXn-­‐  Granular,  brownish-­‐black  crystalline  
deposit  occurring  in  Xssue.  HemaXn  is  the  term  
commonly  used  to  denote  formalin  pigment,  
formed  by  the  acXon  of  acidic  formaldehyde  on  
blood-­‐rich  Xssue,  but  malarial  pigment  and  acid  
hemaXn  are  also  hemaXns.  

40  
 Appendix  low  power  

Loose  ConnecXve  Tissue  

Serosal  Surface  

Mucosal  surface  

Lymphocytes  

41  
Appendix  /  lymphocytes  

Mature  lymphocyte  

Immature  lymphocyte  

Different  stages  of  maturaXon  

42  
 Types  of  Hematoxylin  
 Harris  Hematoxylin  
•  Hematoxylin    
•  Absolute  ethyl  alcohol  
•  Ammonium  aluminum  sulfate  (mordant)  
•  DisXlled  water  
•  Mercuric  oxide  or  sodium  iodate  (oxidizer)    
   
   
 Delafield  Hematoxylin  (oxidaXon  occurs  naturally)  
•  SoluXon  A  
•  Ammonium  Aluminum  sulfate  (mordant)  
•  DisXlled  water  
•  SoluXon  B  
•  Hematoxylin  
•  Alcohol,  95%  

 
•  Glycerol  
   

43  
Tonsil  /  lymphoid  Xssue  

Mature  lymphocyte  

Immature  lymphocyte  

44  
 Types  of  Hematoxylin  
Mayer  Hematoxylin  
•  Hematoxylin  
•  DisXlled  water  
•  Sodium  iodate  (oxidizer)  
•  Ammonium  or  potassium  aluminum  sulfate  (mordant)  
•  Citric  acid  
•  Chloral  hydrate  
   
Ehrlich  Hematoxylin  (oxidaXon  occurs  naturally)  
•  Hematoxylin  
•  Alcohol,  95%  
•  DisXlled  water  
•  Glycerol  
•  Ammonium  or  potassium  aluminum  sulfate  (mordant)  
•  Glacial  aceXc  acid  

45  
 Umbilical  Cord  /  RBC’s  

Smooth  muscle  

Red  Blood  Cells  

Lumen  

46  
 Types  of  Hematoxylin  
Gill  Hematoxylin  
•  DisXlled  water  
•  Ethylene  glycol  
•  Hematoxylin,  anhydrous  
•  Sodium  iodate  (oxidizer)  
•  Aluminum  sulfate  (mordant)  
•  Glacial  aceXc  acid  
•  Note:  Marketed  commercially  in  3  different  strengths  and  mucin,  especially  in  goblet  cells,  will  be  
stained  by  Gill  hematoxylin,  but  not  by  the  other  hematoxylin  soluXons.  
Weigert  Hematoxylin  (Iron  Hematoxylin)  
•  SoluXon  A  
•  Ferric  chloride,  29%  (mordant  and  oxidizer)  
•  DisXlled  water  
•  Hydrochloric  acid,  concentrated  
•                       SoluXon  B  
•  Hematoxylin  
•  Alcohol  
•  Note:  Not  used  in  the  rouXne  H&E  stain  but  used  as  a  nuclear  stain  in  many  of  the  non-­‐rouXne  
techniques  because  it  resists  de-­‐colorizaXon  in  acidic  staining  soluXons.  
   

47  
 Placenta  /  RBC’s  

Red  blood  cells  or  erythrocytes  

Open  chromaXn  

48  
 CelesXne  Blue  
 
•  Ferric  ammonium  sulfate  (mordant)  
•  DisXlled  water  
•  Note:  CelesXne  blue  may  be  subsXtuted  
for  hematoxylin  in  the  H&E  procedure  
and  gives  idenXcal  results.  

49  
 POC  

lymphocyte  

AcXve    Cell  

50  
 DifferenXaXon  
 
Progessively  stained  slides  do  not  require  
differenXaXon  
 
Regressively  stained  slides  are  over  stained  
then  excessive  stain  is    removed    
(differenXaXon)  
 
  51  
 Tonsil  /  knife  mark  

RBC’s  
lymphocytes  

Knife  mark  

52  
 Bluing  
 
Amer  nuclear  staining  with  hematoxylin  soluXons  and  differenXaXon  if  
indicated,  the  secXons  are  blued.  This  is  done  with  soluXons  that  are  
weakly  alkaline.  The  change  in  pH  induced  by  the  bluing  agent  changes  
the  solubility  of  the  dye  lake.  The  aluminum-­‐hematein  complex  is  red  
and  soluble  below  pH  5;  bluing  converts  the  red,  soluble  complex  to  a  
more  desirable  blue  lake  that  is  insoluble  in  the  usual  staining  
soluXons.  
 
•  Dilute  lithium  carbonate  

•  Ammonium  hydroxide  

•  Scot  soluXon  (magnesium  sulfate,  sodium  bicarbonate,  tap  water)  

   

53  
 Plasma  Stains  
 
The  plasma  stains  are  most  frequently  anionic,  or  negaXvely  charged,  dyes  that  combine  with  
very  caXonic,  or  posiXvely  charged,  Xssue  groups.  The  basic  amino  acids,  such  as  arginine,  
hisXdine,  and  lysine,  are  common  sites  for  dye  binding.  
   
Note:  Eosin  is  the  most  widely  used  counter-­‐stain  in  the  rouXne  staining  of  secXons.  The  best  
staining  with  eosin  will  occur  at  a  pH  of  approximately  4.6  to  5.  Used  properly,  at  least  3  
shades  of  pink  can  be  obtained  with  eosin  alone  
 
•  Erythrocytes  (deepest  shade  of  pink)  
•  Collagen  
•  Cytoplasm  of  muscle  or  epithelial  cells  

All  the  above  should  stain  different  intensiXes  of  pink.  Whether  the  collagen  or  muscle/
epithelial  cells  are  the  intermediate  shade  of  pink  is  determined  by  choice  of  fixaXve,  
duraXon  of  fixaXon,  heat  during  processing,  stain  formulaXon,  differenXaXon  step  and  the  
nature  of  the  collagen  itself.  

54  
 Colon  Polyp  
Red  blood  cells  
lymphocytes  

Glandular  cells  

55  
 Eosin  
Eosin  Counter-­‐stain  
•  Eosin  Y  (1%  aqueous  soluXon)  
•  Ethyl  alcohol,  95%  
•  AceXc  acid,  glacial  
   
Eosin-­‐Phloxine  B  Counter-­‐stain  
•  Eosin  Y  (1%  aqueous  soluXon)  
•  Phloxine  B  (1%  aqueous  soluXon)  
•  Alcohol,  95%  
•  AceXc  acid,  glacial  
•  Note:  Some  laboratories  prefer  an  eosin-­‐phloxine  B  soluXon,  
because  the  pink  shades  are  more  vivid  but  must  be  careful  
not  to  over-­‐stain.  

56  
 Colon  Polyp  /  Eosinophil  

lymphocyte  

Glandular  cells  

Eosinophil  

57  
H&E  Progressive  Stain  
 
1.  Xylene,  3  changes        2  minutes  each  
2.  Absolute  alcohol          10  dips  
3.  Alcohol,  95%,  2  changes        10  dips  each  
4.  Tap  water          Rinse  unXl  water  
runs  off            
 evenly  
5.  Hematoxylin,  Mayer        15  minutes  
6.  Or  acidified  Harris        1-­‐3  minutes  
7.  Tap  water,  2  changes        10  dips  each  
8.  Ammonia  water,  0.25%  or        unXl  blue  
9.  Lithium  carbonate,  0.5%  
10.  Tap  water,  2  changes        10  dips  each  
11.  Eosin            10-­‐20  dips  
12.  Or  Eosin  –  phloxine        1-­‐3  minutes  
13.  Alcohol,  70%          10-­‐15  dips  
14.  Alcohol,  95%          10-­‐15  dips  
15.  Absolute  alcohol,  3  changes        10-­‐15  dips  each  
16.  Xylene,  3  changes        10-­‐15  dips  each  
•  Let  slides  remain  in  last  container  of  xylene  unXl  a  coverslip  is  applied.  

58  
 H&E  Regressive  Stain  
 
1.  Xylene,  3  changes        2  minutes  each  
2.  Absolute  alcohol        10  dips    
3.  Alcohol,  95%,  2  changes        10  dips  each  
4.  Tap  water        rinse  unXl  water  runs  off    
             evenly  
5.  Hematoxylin,  Delafield,  Ehrlich,      10-­‐15  minutes  
           Or  Harris  without  acid  
6.                            Tap  water,  2  changes        10  dips  each  
7.                            Hydrochloric  acid  1%  in  70%  alcohol      5-­‐10  dips  
8.                            Running  water        wash  well  
9.                            Ammonia  water,  0.25%        unXl  blue  
                                   Or  lithium  carbonate,  0.5%  
10.                        Tap  water,  2  changes        10  dips  each  
11.                          Eosin          10-­‐20  dips  
                                     Or  eosin-­‐phloxine        1-­‐3  minutes  
12.                          Alcohol,  70%        10-­‐15  dips  
13.                          Alcohol,  95%        10-­‐15  dips  
14.                        Absolute  alcohol  3  changes      10-­‐15  dips  each  
15.                        Xylene,  3  changes        10-­‐15  dips  each  
16.  Let  slides  remain  in  last  container  of  xylene  unXl  a  coverslip  is  applied.  

                   RESULTS  
•  Nuclei      Blue  
•  Erythrocytes  and  eosinophilic  granules  Bright  pink  to  red  
•  Cytoplasm  and  other  Xssue  elements  Pink  shades  
•     

59  
Cervical  Bx  with  HPV  effect  

The  Koilocyte  displays  a  large  perinuclear  halo  with  irregular  clear-­‐cut  edges  (HPV)  

Koilocyte      

60  
Cervical  Bx  High  grade    

Irregular  cell  with  nucleoi  

Irregular  nuclear  border  

61  
Cervical  Bx  High  grade  

nucleoi  

Irrgular  nuclear  border  

Intranuclear  cytoplasmic    inclusion  

Clumpy  chromaXn  

62  
 Pap  smear  low  grade  
with  HPV  

Normal  intermediate  cell  

Perinuclear  halo  

63  
 Pap/low  grade  with  HPV  

KeraXnized  cytoplasm  

Perinuclear  halo  

64  
 Pap  smear  high  grade  

HyperchromaXc  nuclie  

Increased  N/C  raXo  

65  
 Pap  smear  posiXve  
adenocarcinoma  

Adenocarcinoma  cells  

Normal  squamous  cell  

66  
 Hints  to  Help  Achieve  Good  
H&E  Staining  
 
•   Microscopically  check  control  slide  (small  intesXne)  prior  to  running  
paXent  samples  
•  Do  not  allow  secXons  to  dry  at  any  point  during  staining  
•  Keep  soluXons  covered  while  not  in  use.  Make  sure  soluXons  completely  
cover  slides.  If  any  precipitate  is  noted  at  the  top  of  the  hematoxylin  
container,  filter  the  soluXon  into  a  clean  dry  container.l  
•  Develop  a  rouXne  schedule  for  changing  soluXons  based  on  the  number  of  
slides  stained  each  day.  Record  and  document.  
•  Amer  applying  the  bluing  soluXon  wash  the  secXons  very  well;  any  carry  
over  of  the  bluing  soluXon  will  change  the  pH  of  the  eosin  and  cytoplasmic  
staining  will  be  lacking.  
•  Do  not  pass  the  slides  through  the  dehydraXng  soluXons  too  quickly,  
because  dehydraXng  soluXons  also  serve  to  differenXate.  Keep  in  mind  
that  the  more  dilute  the  alcohol,  the  more  eosin  that  will  be  removed.  
 

67  
Hints  to  Help  Achieve  Good  
H&E  Staining  
•  Tissues  that  have  been  fixed  for  longer  than  normal  may  require  increased  staining  Xmes.  
•  Staining  Xmes  also  may  need  to  be  adjusted  according  to  the  fixaXve  used,  the  Xme  in  
hematoxylin  may  need  to  be  increased  amer  fixaXon  in  Helly,  Zenker,  or  B-­‐5  fixaXves,  and  the  
Xme  in  eosin  will  frequently  need  to  be  decreased.  
•  If  using  a  xylene  subsXtute,  the  manufacturer’s  recommendaXons  must  be  closely  followed.  
•  Daily  check  and  record  the  pH  of  the  running  tap  water.  Some  tap  water  may  not  be  acceptable  
before  or  amer  hematoxylin.  Iron,  sulfur,  and  chlorine  will  produce  weak  nuclear  staining.  
Chlorine  content,  which  varies  seasonally,  will  also  cause  staining  variability.  Highly  alkaline  or  
hard  water  may  serve  as  an  excellent  bluing  agent  but  may  create  dark  nuclear  or  background  
staining.  
•  Remember  that  what  appears  to  be  a  staining  problem  is  not  always  a  staining  problem.  If  the  
problem  cannot  be  idenXfied  easily  as  a  staining  problem,  cut  and  stain  secXons  from  a  
previous  day’s  workload  in  which  the  staining  was  excellent.  If  the  staining  is  sXll  excellent  on  
the  previous  material,  then  the  source  of  the  problem  must  be  in  some  other  area.  The  duraXon  
of  fixaXon,  the  use  of  heat  during  processing,  and  the  possible  carryover  of  formalin  or  water  
into  the  clearing  and  infiltraXon  reagents  are  areas  that  should  be  examined  when  an  apparent  
staining  problem  is  proved  not  to  be  so.  
   

68  
Restoring  Tissue  Basophilia  
 
Staining  properXes  of  markedly  over-­‐decalcified  bony  Xssues  cannot  be  restored,  so  proper  
iniXal  decalcificaXon  is  very  important.  
 
Method  I  
•  Place  deparaffinized  slides  from  Xssue  overexposed  to  Bouin  soluXon  in  5%  aqueous  
lithium  carbonate  soluXon  for  1  hour.  
•  Wash  in  running  tap  water  for  10  minutes,  and  stain  using  desired  method.  
             Method  II  
•  Place  deparaffinized  slides  in  a  5%  aqueous  sodium  bicarbonate  soluXon  for  3  hours  (4  
hours  for  Xssues  overexposed  to  Zenker  soluXon).  
•  Wash  in  tap  water  for  5  minutes,  and  stain  using  desired  method.  
           Method  III  
•  Place  deparaffinized  slides  in  5%  aqueous  periodic  acid  for  30  minutes.  
•  Rinse  in  3  changes  of  disXlled  water,  and  stain  using  desired  method.  
   

69  
 Frozen  SecXon  Staining  
 
•  Cut  the  frozen  secXon  and  fix  in  37%  to  40%  formaldehyde  for  20  seconds  (I  prefer  using  
alcoholic  formalin)  
•  Rinse  the  secXon  very  well  in  at  least  3  changes  of  tap  water  (if  using  alcoholic  formalin  
one  water  rinse)  
•  Stain  in  Harris  hematoxylin  with  aceXc  acid  for  1  to  1  ½  minutes  
•  Rinse  in  2  two  changes  of  tap  water  
•  Place  slide  in  0.25%  ammonia  water  or  another  bluing  agent,  and  leave  unXl  blue.  
•  Rinse  in  2  changes  of  tap  water  
•  Stain  in  eosin  with  15  to  20  dips  or  unXl  the  desired  intensity  is  achieved.  
•  Dehydrate  with  95%  alcohol  and  absolute  alcohol  –  10  dips  in  2  changes  of  each  alcohol  
•  Clear  the  secXons  with  xylene  –  10  dips  in  3  changes  
•  Mount  with  syntheXc  resin  
•  Results:  
•  Nuclei          Blue  
•  Cytoplasm  and  other  Xssue  elements    Shades  of  pink  
   

70  
 Notes  on  Frozen  SecXon  
Staining  
 
•  Fix  cut  secXons  immediately;  do  not  allow  the  
slides  to  air-­‐dry  or  morphologic  preservaXon  will  
be  poor  
•  Can  stain  with  rapid  H&E  stain  or  use  
metachromaXc  dyes  such  as  toluidine  blue  O  or  
polychrome  soluXons  (purchased  commercially)  
•  SoluXons  should  be  changed  on  a  regular  basis  
depending  on  usage  
•  Alcoholic  formalin  may  be  used  instead  of  
concentrated  formaldehyde  

71  
 TROUBLESHOOTING  THE  
H&E  STAIN  
 
Incomplete  Deparaffiniza2on  
•  Dry  secXons  properly  before  beginning  deparaffinizaXon;  if  improper  drying  is  the  cause,  
slides  can  be  treated  with  absolute  alcohol  to  remove  the  water,  and  then  re-­‐treated  with  
absolute  alcohol  to  remove  the  water,  and  then  re-­‐treated  with  xylene  to  remove  the  
paraffin;  if  incomplete  drying  is  severe,  the  secXons  may  loosen  from  the  slides  
•  Allow  sufficient  Xme  in  xylene  for  complete  deparaffinizaXon;  if  this  is  the  cause,  return  to  
xylene  for  a  longer  Xme  
•  Avoid  contaminated  xylene;  change  the  soluXon  if  necessary  
•  If  the  slides  have  been  stained,  decolorize  and  restain  
   
Nuclear  staining  is  not  crisp  
•  Fix  Xssue  specimen  completely  
•  Dehydrate  and  clear  Xssues  completely  before  infiltraXng  with  paraffin  
•  Do  not  use  heat  on  the  processor  except  for  the  paraffins  
•  Do  not  leave  Xssue  in  melted  paraffin  for  a  prolonged  period  
•  Dry  microscopic  slides  at  the  correct  temperature  (60-­‐70  degrees  C)  and  for  the  shortest  Xme  
possible  that  ensures  complete  drying  

72  
 TroubleshooXng  H&E  stain  
Pale  Nuclear  staining  
•  Not  leaving  in  hematoxylin  long  enough  
•  Staining  with  over  oxidized  or  depleted  hematoxylin  
•  Over  differenXaXng  the  hematoxylin  
•  Note:  pale  nuclei  in  bone  secXons  may  result  from  over-­‐decalcificaXon  
   
   
Dark  Nuclear  Staining  
•  SecXons  lem  too  long  in  hematoxylin  
•  SecXons  too  thick  
•  DifferenXaXon  step  too  short  
   
Red  or  Red-­‐Brown  Nuclei  
•  Ensure  that  secXons  are  blued  properly;  it  is  not  possible  to  over-­‐blue  the  secXons  
•  Check  oxidaXon  status  of  hematoxylin  as  given  in  secXons  following  “Delafield  
hematoxylin”  

73  
 TroubleshooXng  H&E  Stain  
Pale  Cytoplasmic  staining  
•  Check  eosin  soluXon  pH;  adjust  with  aceXc  acid  if  necessary  
•  Completely  remove  bluing  reagent  before  transferring  the  slide  to  eosin  
•  Do  not  allow  stained  slides  to  stand  in  the  lower  concentraXons  of  
alcohols  amer  the  eosin;  the  more  water  in  the  alcohol,  the  more  eosin  
that  will  be  removed  
•  Ensure  that  secXons  are  not  too  thin  
   
Dark  cytoplasmic  staining  
•  Avoid  over  concentrated  eosin  soluXon,  especially  if  phloxine  is  present;  if  
necessary,  dilute  the  eosin  soluXon  
•  Do  not  leave  secXon  in  eosin  too  long  
•  Allow  sufficient  Xme  in  dehydraXng  soluXons,  especially  70%  alcohol,  to  
allow  good  eosin  differenXaXon  
•  Check  secXon  for  proper  thickness  
   

74  
 Eosin  not  properly  
differenXated    
If  3  shades  of  eosin  are  not  apparent  
 
•  Timely  and  complete  fixaXon  
•  Good  dehydraXon  and  clearing  during  processing  
•  Eosin-­‐stained  secXon  remains  in  the  lower  
diluXons  of  alcohol  for  proper  differenXaXon;  
adequate  Xme  in  70%  alcohol  will  give  the  best  
differenXaXon  of  the  eosin  
•  Eosin  is  at  the  correct  pH  
   
 

75  
 TroubleshooXng  H&E  Stain  
Blue-­‐Black  Precipitate  on  top  of  sec2ons  
•  Filter  hematoxylin  
•  Hazy  or  Milky  water  and  slides  
•  When  the  slides  and  water  turn  milky  following  the  rehydraXng  alcohols,  it  indicates  the  
presence  of  xylene  on  the  slides  
•  When  slides  appear  hazy  or  milky  in  the  last  xylene  used  in  clearing,  it  indicates  that  
water  is  sXll  present  on  the  slides  and  that  dehydraXon  is  not  complete.  
   
Uneven  H&E  staining  
•  May  be  caused  by  water  or  fixaXve  in  the  infiltraXng  paraffin,  or  by  contaminaXon  of  
reagents  in  closed  Xssue  processors  because  of  equipment  malfuncXon  or  absorpXon  of  
atmospheric  water  by  the  dehydraXng  alcohols  on  the  open  processors.  
•  Use  toluene  instead  of  xylene  in  areas  of  high  humidity  if  using  open  processors;  toluene  
is  more  water  tolerant  than  xylene  
•  Check  equipment  for  malfuncXon  
   

76  
 TroubleshooXng  H&E  Stain  
Dark  Basophilic  staining  of  nuclei  and  cytoplasm,  
especially  around  2ssue  edges  
•  Laser  and  electro-­‐cautery  techniques  denature  
macromolecules  and  produce  heat  arXfact,  generally  
marked  by  dark  basophilic  staining  in  nuclei  and  
cytoplasm.  There  is  no  remedy  for  this  arXfact  
Poor  contrast  between  nucleus  and  cytoplasm  
•  The  nucleus  is  too  pale  to  contrast  well  with  the  
cytoplasm  
•  The  cytoplasm  is  over-­‐stained  and  masks  the  nuclei  
•  The  nuclear  stain  is  too  dark  for  the  cytoplasmic  stain  
•  The  cytoplasmic  stain  is  too  pale  for  the  nuclear  stain  

77  
 Nucleic  Acid  Stains  
 
Feulgen  Reac2on  
•  For  the  demonstraXon  of  DNA  
•  Reagents  
•  Hydrochloric  Acid  
•  Schiff  Reagent  
•  Sulfurous  Acid  
•  Results:  DNA    Reddish  purple  
•                                                       Cytoplasm            Light  green  

Methyl  Green-­‐Pyronin  Y  
•  To  differenXate  between  DNA  and  RNA  
•  Reagents  
•  Glacial  aceXc  acid  
•  Sodium  Acetate  
•  Methyl  green  dye  
•  Results:  DNA  (green  to  blue-­‐green),  RNA  (Red  to  rose),  Goblet  cells  (mint  green,  Background  (pale  pink  to  
colorless),  Immunoblast  and  plasma  cell  cytoplasm  (intense  red),  Nuclei  (green  to  blue-­‐green)  

78  
 CPT  Codes  
•  Services  88300  through  88309  include  
accession,  examinaXon,  and  reporXng.    
 

79  
THE  END  
Lung  Xssue  

artery  

GI  Xssue  

80