Histological Specimen Preparation

1. Tissue Processing
Dr. Aisha Khatoon
DVM(Gold Medal), M. Phil, Ph. D
Or Infiltrartion
⦿Tissue specimens received in the surgical pathology laboratory
have a request form that lists the patient information and
history along with a description of the site of origin

⦿ Thespecimens are accessioned by giving them a number

that will identify each specimen for each patient.
Preliminary Steps for Optimal Processing

Steps leading to the processing stage are crucial for obtaining morphological and histochemical information from
the specimen.

Fixation Decalcification Grossing Enclosing

8
Preliminary Steps: Fixation

• Fixation is a critical step in the preparation of histological

sections. If it is not carried out under optimal conditions or if
fixation is delayed, a tissue specimen can be irreversibly
damaged compromising morphological and histochemical
information.

The optimal time for fixation will vary between

•
fixatives, tissue type and size. Dense or fatty tissues
usually require more time to be fully fixed.

• Most frequently the routine fixative will be neutral buffered

formalin.

Discussed in detail in earlier lectures.

9
Preliminary Steps: Decalcification

• Bone and other calcified specimens must be

decalcified prior to processing and paraffin
infiltration.
• Once the mineral has been removed, a standard
processing schedule can be used.
• Most of the acid decalcifier should be washed away
before processing to avoid contaminating the
processing reagents.
• Despite complete decalcification, bone (particularly
compact bone), will contain dense areas that
require thorough processing. In such case choosing
a longer schedule is advised.

10
⦿The Process of removing calcium salts from the tissue and making them suitable for
sectioning.
⦿ Some tissues contain calcium deposits which are extremely firm and which will not section
properly with paraffin embedding owing to the difference in densities between calcium and
paraffin.
⦿ Bone specimens are the most likely type here, but other tissues may contain calcified areas as
well
⦿ This calcium must be removed prior to embedding to allow sectioning.
⦿ A variety of agents or techniques have been used to decalcify tissue and and none of them
work perfectly.
◾S t r o n g Mineral acids,
◾We a k organic acids,
◾ EDTA as Chelating agent
• Specimens should be decalcified in hydrochloric acid/formic acid working
solution 20 times their volume.
• Change to fresh solution each day until decalcification is complete.
 It may take 24 hours up to days or months depending on size of the specimens.
 Once the decalcification is complete, rinse specimens in water briefly and transfer to ammonia
solution to neutralize acids left in specimens for 30 minutes.
• Wash specimens in running tap water thoroughly up to 24 hours.
• Routine paraffin embedding.
 Other Techniques for Increasing the Efficiency of
Decalcification

1. Sonication used with EDTA has been successfully used to accelerate decalcification of
trephine specimens for subsequent molecular analysis.

2. Microwave treatment has been used with hydrochloric acid decalcifiers but the raised
temperature may damage morphology and cause staining artefacts

3. Ion-exchange resins have been incorporated into some decalcification protocols. They are
added to the container holding the decalcifier and take up the ionized calcium maintaining the
effectiveness of the acid. If acid decalcifiers are used in adequate volumes and replaced
regularly the use of such resins is probably unnecessary

4. Electrolytic decalcification in which the bone is placed in acid decalcifier and attached to an
electrode through which current is applied is a technique that has not found wide acceptance
because of the potential to cause heat damage to the specimen
⦿ Aqueous nitric acid

⦿ Nitric acid –formaldehyde(recommended for urgent biopsies)

⦿ Perenyi’s fluid—Excellent cytological preservation
⦿ Hydrochloric acids
⦿ Nitric
and
⦿ Hydrochloric acids
◾ rapid
◾ damage cellular morphology,
◾ soare not recommended for delicate tissues such as bone
marrow.
 Table 1: Mineral acid decalcifiers

Decalcifier Formula Comment

Nitric acid 5% in distilled water Rapid in action, exceeding end-point will impair staining.
Perenyi’s fluid 10% nitric acid 40ml A traditional decalcifier that decalcifies more slowly than aqueous nitric
1882) 0.5% chromic acid 30ml acid. Quite rapid in action, exceeding end-point will impair staining.
Absolute alcohol 30ml
Hydrochloric acid 5-10% in distilled water Formalin should be washed from specimen before placing in HCl to avoid
the formation of bis-chloromethyl ether (a carcinogen). Rapid in action,
exceeding end-point will impair staining.
Von Ebner’s Sodium chloride saturated soln. Rapid in action, exceeding end-point will impair staining.
solution 50ml
Distilled water 42ml
Hydrochloric acid 8ml
⦿Acetic and Formic acid are better suited to bone marrow, since they are
not as harsh
⦿However, they act more slowly on dense cortical bone.

⦿Formic acid in a 10% concentration is the best all-around decalcifier.

⦿Some commercial solutions are available that combine formic acid with
formalin to fix and decalcify tissues at the same time.
 Table 2: Weak acid decalcifiers

Decalcifier Formula Comment

Formic acid 10% in distilled water A simple effective decalcifier.
Evans and Krajian Formic acid 25ml An effective formic acid
Sodium citrate 10g decalcifier buffered with
Distilled water 75ml citrate.
Kristensen Formic acid 18ml An effective formic acid
Sodium formate 3.5g decalcifier buffered with
Distilled water 82ml formate
Gooding and Stewart Formic acid 5-25ml A formic acid decalcifier with
40%formaldehyde 5ml added formalin, claimed to fix
Distilled water 75ml and decalcify.
⦿Most used is EDTA which as ability to bind calcium forming
non-ionized soluble complex
⦿ EDTA works best on cancerous bone

⦿ Agent of choice for electron microscopy

⦿EDTA can remove calcium and is not harsh (it is

not an acid) BUT
◾it penetrates tissue poorly
◾works slowly
◾expensive in large amounts.
 Table 3: Chelating agents

Decalcifier Formula Comment

Neutral EDTA EDTA disodium salt 250g Acts slowly but causes
Distilled water 1750ml little tissue damage.
Bring to pH 7.0 by adding Conventional stains are
sodium hydroxide (about largely unaffected.
25g will be needed).
⦿ Concentration of active reagent
It must be remembered that the concentration of active agent will be depleted as it combines
with calcium and so it is wise to use a large volume of decalcifier and renew it several times
during the decalcification process

⦿T
emperature
Increased temperature will speed up the decalcification rate but will also increase the rate of
tissue damage so must be employed with great care

⦿ Agitation
Gentle agitation may increase the rate slightly

⦿ Density of bone
⦿ X-ray (the most accurate way)
⦿ Chemical testing (accurate)
⦿ Physical testing (less accurate and potentially
damage of specimen)
Figure : An X-ray series following the process of decalcification of a femoral
head with formic acid/citrate decalcifier. The radiographs were produced using a
Hewlett-Packard Faxitron® and allow the process to be accurately followed and
the endpoint to be properly identified.
⦿ Insert a pipette into the decalcifying solution containing the
specimen.
⦿ Withdraw approximately 5 ml of the hydrochloric acid/formic
acid decalcification solution from under the specimen and
place it in a test tube.
⦿ Add approximately 10 ml of the ammonium
hydroxide/ammonium oxalate working solution, mix well and
let stand overnight.
⦿ Decalcification is complete when no precipitate is observed
on two consecutive days of testing.
Repeat this test every two or three days.
⦿The Physical tests include bending the specimen or inserting a pin,
razor, or scalpel directly into the tissue.

⦿The disadvantage of inserting a pin, razor, or scalpel is the

introduction of tears and pinhole artifacts.

⦿Slightly bending the specimen is safer and less disruptive but will not
conclusively determine if all calcium salts have been removed.

⦿After checking for rigidity, wash thoroughly prior to processing.

 Preliminary Steps: Grossing

• For optimal fixation and subsequent high-quality processing the dimensions of tissue specimens are
important. Ideally the thickness should not exceed 5mm and the specimen should fit into a histology
cassette without distortion.
• It is possible to “over-process” a small and delicate specimen or “under-process” large, dense
specimens making them very difficult, if not possible, to section on a microtome.
• Processing times are also different for different tissue types: some tissues are penetrated by reagents
relatively easily (kidney, lung) while others are much more resistant (cervix, muscle) and require more
time, i.e. different schedule.

 Tissues removed from the body for diagnosis arrive in the Pathology Department and are examined by a
pathologist, pathology assistant, or pathology resident.
 Gross examination consists of describing the specimen and placing all or parts of it into a small plastic
cassette which holds the tissue while it is being processed to a paraffin block. Initially, the cassettes are placed
11
into
a fixative
Preliminary Steps: Enclosing Specimen

• Cassettes hold and protect the specimen while it undergoes processing.

• Cassettes chosen must be completely resistant to the solvents and heat used in processing. They must not
distort during use so that there is no chance that the specimen will escape into the processing reagents.
• Perforated cassette surfaces must allow for adequate fluid exchange and proper drainage.
• Small specimens can be protected by wrapping them in fine, lint-free papers such as bio-wraps, placing
them in fine-mesh biopsy bags or „sandwiched‟ by using biopsy pads.

12
What is Tissue Processing?

• Tissue processing is a procedure of removing water from cells and replacing it

with a medium which solidifies allowing thin sections to be cut on a microtome.

• The aim of tissue processing is to embed the tissue in a solid medium firm
enough to support the tissue and give it sufficient rigidity to enable thin sections
to be cut , and yet soft enough not to damage the knife or tissue.

• Once tissue is properly fixed it goes through a process which involves the
following steps:
• Dehydration
• Clearing
• Infiltration & Embedding
Stages of processing tissu es for histopathological examination

1 Dehydration.
2 Clearing/Dealcoholization
3Impregnation/ wax infiltration
4- Embedding/ Block formation
5- Sectioning/ cutting:
Trimming
Sectioning– Ribbon
6. Floating water bath- straightening
7- Oven?
8 Staining
9 Mounting
Processing Methods

Manual Processing
• Slow, most labor intensive method since transfer of specimens or changing reagents is done by hand. With
an advance of automation this method is almost obsolete.
Automated Processing
• Specimen-transfer or “dip and dunk” processors: instruments which transfer cassettes from station to
station in a rotary or linear configuration.
• Fluid-transfer or “enclosed” instruments hold the specimens in a process chamber or retort and the
reagents are pumped in and out during processing.
• Microwave assisted processing: might require manual transfer of specimen or reagents, it accelerates
processing by heating reagents.7
Tissue Processing - Overview

• “Tissue processing” describes the steps required to take animal or human tissue from fixation to
the state of complete infiltration with a histological paraffin.
• Subsequently, the processed tissue is made into a paraffin block so it can be sectioned on the
microtome.

Alcohol replaces water in

Step 1: Alcohol all cells

Step 2: Xylene Xylene dissolves alcohol

Paraffin displaces xylene.

Step 3: Paraffin Specimen is now ready to
be embedded.

Specimen
3
Step 1: Dehydration

Alcohol replaces water in

Alcohol
all cells

Specimen

• Since paraffin is hydrophobic (immiscible i.e. not mixable with water), water inside a specimen must be
removed before it can be infiltrated with paraffin. This process is carried out by immersing specimens in
a series of alcohol.
• Alcohol progressively replaces water in all the cells of the specimen.
• A series of increasing (typically from 70% to 100%) alcohol concentrations are used to avoid excessive
distortion of the tissue.

4
Step 2: Clearing

Xylene Xylene dissolves alcohol

Specimen

• Since alcohols and paraffins are not miscible, an intermediate solvent that is fully miscible with both (such
as xylene), must be used.
• This solvent displaces the alcohol in the tissue through the process called “clearing”.
• “Clearing” relates to how clearing agents impart an optical clarity or transparency to the tissue due to their
relatively high refractive index.
• Another important role of the clearing agent is to remove a substantial amount of fat from the tissue
which otherwise presents a barrier to paraffin infiltration.
• To make sure that all traces of alcohols are removed from tissues being processed, multiple changes of
fresh xylene, clear of carried-over alcohol, are required.

5
Step 3: Infiltration

Paraffin Paraffin displaces xylene

Specimen

• The specimen can now be infiltrated with paraffin. Molten paraffin infiltrates tissues and when cooled
solidifies to a consistency that allows sectioning on a microtome.
• The amount of structural support given by solidified paraffin can be regulated by choosing different
paraffin formulations.
• Multiple changes of histological paraffin are required to completely displace the clearing agent.
• Paraffin infiltration is greatly enhanced by vacuum.

6
 ⦿ Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated
with paraffin.

⦿ First, the water from the tissues must be removed by dehydration

⦿ This is usually done with a series of alcohols, say 70% to 95% to
100%.
⦿ Sometimes the first step is a mixture of formalin and alcohol.
⦿ Other dehydrants can be used but have major disadvantages.

◾ Acetone is very fast, but a fire hazard, so is safe only for small, hand-
processed sets of tissues.
◾ Dioxane can be used without clearing, but has toxic
fumes
⦿ Alcohols –
Ethanol
Methanol
Isopropanol

⦿ Normal and tertiary glycol-

ethers butanols
Ethoxyethanol,polyethylene glycols

⦿ Other dehydrants-----
Acetone
Phenol
Beechwood Cresolate
Aniline
⦿ Ethanol

-clear, colorless, flammable

-hydrophillic
Advantages -------non toxic,reliable
Disadvantage------expensive,tissue shrinkage
⦿ Supplied
as 99.85% ethanol
⦿Anhydrous cupric sulphate added to final ethanol
scavanges any water present.
⦿ Duration of dehydration should be kept to the minimum consistent with the
tissues being processed

⦿ Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol

⦿ Blocks 5 mm thick require up to 90 minutes or longer in each change

⦿ Tissues may be held and stored indefinitely in 70% ethanol without harm
SCHEDULE FOR DEHYDRATION WITH ALCOHOL
30%  2hrs

50%  2hrs

70%  2hrs

80%  1hr

95%  1hr

95%  2hr

Abs. Alcohol I30mint

Abs. Alcohol II  30mint.

 CLEARING
 Removal of the dehydrant with a substance that will be miscible with the
embedding medium (paraffin)

 The commonest clearing agent is xylene

 Toluene works well, and is more tolerant of small amounts of water left in the
tissues, but is 3 times more expensive than xylene

 Chloroform used to be used, but is a health hazard, and is slow

 Methyl salicylate is rarely used because it is expensive, but it smells nice (it is
oil of wintergreen).
⦿Replacing the dehydrating fluid with a fluid that is totally miscible with
both the dehydrating fluid and the embedding medium

⦿Choice of a clearing agent depends upon the following:

- The type of tissues to be processed, and The type of processing to be
undertaken
- The processor system to be used.
-Intended processing conditions such as temperature, vacuum
and pressure.
- Safety factors.
- Cost and convenience.
- Speedy removal of dehydrating agent .
- Ease of removal by molten paraffin wax .
- Minimal tissue damage .
⦿ Chloroform – tolerant, no effect on RI

⦿Xylene,
Benzene, toluene – rapid, intolerant,
flammable, affects RI

⦿ Esters—n butyl acetate----xylene substitute

⦿ cedar wood oil – tolerant, expensive

⦿ When xylene has completely replaced the
alcohol in the tissue, the specimen is ready to be
infiltrated with paraffin.
⦿It is removed from the xylene and placed in a
dish of embedding paraffin, and the dish is put in
a constant temperature of about 60 °C
⦿ The exact temperature depend upon melting
point of the paraffin used.
⦿During the course of several hours the specimen
is changed to two or three successive dishes of
paraffin so that all xylene in tissue is replaced by
paraffin
⦿Finally, the tissue is infiltrated with the embedding agent, almost
always paraffin.
⦿ Paraffins can be purchased that differ in melting point, for various
hardnesses, depending upon the way the histotechnologist likes
them and upon the climate (warm vs. cold).
⦿Wax hardness (viscosity) depends upon the molecular weight of
the components and the ambient temperature.
⦿ High molecular weight mixtures melt at higher temperatures than
waxes comprised of lower molecular weight fractions.
⦿ Paraffin wax is traditionally marketed by its melting points which
range from 39°C to 68°C.
 VARIOUS TYPES OF EMBEDDING MEDIUM

1.) Paraffin wax – Paraffin is solid at room temperature. The melting point of
paraffin ranges from 40-60°C. For tropical countries hard wax having a melting
point of 58-60°C is suitable. This is the most commonly used tissue embedding
medium.

2.) Paraplast – This is a mixture of purified paraffin and plastic. It is easy to make
the serial sections of 4 microns with this embedding medium due to its elasticity.

3.) Gelatin – Water soluble and so dehydration and Clearing is not necessary. It is
used for delicate tissues and for the frozen sections. It has low melting point.
4.) Ester wax – It is harder than paraffin but has a low melting point of 46-
48°C. It is soluble in alcohol so there is no need for the clearing step when
using this tissue embedding medium.

5.) Tissue mate – It is the mixture of paraffin and rubber and has similar
properties as that of Paraplast. The serial sections can easily be obtained
using this embedding medium.

6.) Water-soluble waxes – These are Polyethylene glycol, having a melting

point of 38-42°C. They cause less shrinkage than paraffin and
no dehydration and clearing is needed. They do not make the tissues brittle &
give better support than paraffin. They are used for demonstration of lipid
and enzymes.
7.) Celloidin – This is purified nitrocellulose and is used for the hard and
fragile specimens. Large specimens can easily be sectioned using this
embedding medium. It takes 2-3 weeks to impregnate the tissues with
Celloidin.
⦿ Paraffin wax embedding
⦿ Water soluble waxes embedding
⦿ Celloidin embedding
⦿ Double embedding
⦿ Gelatin embedding
⦿ Ester wax embedding
There are four main mould systems and associated embedding
protocols presently in use :

1 Traditional methods using paper boats.

2Leuckart or Dimmock embedding irons or metal containers.

3- The Peel-a-way system using disposable plastic moulds and

4- Systems using embedding rings or cassette-bases which

become an integral part of the block and serve as the block
holder in the microtome.
Tissue processing Embedding moulds:

(A) paper boat;

(B) metal bot mould;

(C) Leuckhart or Dimmock embedding mould;

(D) Peel-a-way disposable mould;

(E) base mould used with embedding ring (F)

(G) Tissue Cassette

General Embedding Procedure
1- Open the tissue cassette, check against worksheet entry to ensure
the correct number of tissue pieces are present.

2- Select the mould, there should be sufficient room for the tissue with
allowance for at least a 2 mm surrounding margin of wax.

3- Fill the mould with paraffin wax.

4 Using warm forceps select the tissue, taking care that it does not cool
in the air; at the same time, correct orientation of tissue in a mould is
the most important step in embedding. Incorrect placement of tissues
may result in diagnostically important tissue elements being missed or
damaged during microtomy.

5Insert the identifying label or place the labeled embedding ring or

cassette base onto the mould.

6 Cool the block on the cold plate, or carefully submerge it under

water when a thin skin has formed over the wax surface.

7 Remove the block from the mould

.
8 Cross check block, label and worksheet.
tek
 MICROTOMY
(Sectioning of Tissues)

A section is a thin transparent slice of body tissue stained and covered by a cover
slip on a glass slide
⦿ A microtome is a mechanical
instrument used to cut biological specimens into very
thin segments for microscopic examination

⦿ Most microtome use a steel blade and are used to

prepare sections of animal or plant tissues for histology.
Types of Microtomes
An instrument used for cutting sections .
1) Rotary Microtome
For paraffin sections and cryostat sections.

2) Sliding and Base sledge

•Large and heavy instrument.
•Used for nitrocellulose and plastic sections.
•Slow and expensive.
•Unexcelled for large and hard object such as eyes, bone and cartilage.

3) Clinical (Freezing )microtome.

•Used for fixed or unfixed unembedded tissue.
•Knife more horizontally across the surface of object.
•Give immediate , quick and cheap diagnostic .Highly recommended for cutting unfrozen
fresh or unfixed tissue.
4) Cambridge Rocking Microtome.
•Small and light weight instrument .
•Knife is fixed but object moves in a circle against the knife.
•Suitable for class work, not suitable for large blocks of hard materials.

5) Ultra thin sectioning microtome.

•Designed for electronic microscopy.
•Give section of 0.005- 0.1µ thickness.
•Not commonly found in student lab.

6) Fast scientific microtome.

•Small, inexpensive and portable instrument.
•Cuts section of about 25 µ with razor blade.
Slides:
a) Size 7.5 × 2.5 cm
b) Cover glasses
i) Shape  circle (9-21mm), squares( 1.3cm), rectangles(22mm -24×30-60mm)
ii) Thickness:
 No. (0-1) (0.09- 0.15mm thick) used for oil immersion work.
 No. (2) (0.20mm thick) commonly used for whole mounts not to be examined at
high day power objections.
 No. (3) (0.30 – 0.35mm) inlarge whole mounts.
Preparations of Slides:
Cleaning of slides.
Adhesive mixtures Meyer’s egg albumin. (50ml albumin+ 50ml glycerol) Mix
well
i) Egg albumin and water.
ii)Gelatin.
iii)Blood serum.
⦿Turn on the water bath and check that the
temperature is 35-37ºC.
⦿ Use fresh deionized water (DEPC treated water must be used
if in situ hybridization will be performed on the sections).

⦿ Blocks to be sectioned are placed face down on an ice block

or heat sink for 10 minutes.
⦿ Place a fresh blade on the microtome
⦿ Insert the block into the microtome chuck so wax block faces
block faces the blade and is aligned in the vertical plane. Set
the dial to cut 4-10 µm sections.
⦿ The blade should be angled 4-6º.
⦿ Face the block by cutting it down to the desired tissue plane and discard the paraffin
ribbon.
⦿If the block is ribboning well then cut another four sections and pick them up with
forceps or fine paint brush and float them on the surface of the 37ºC water
bath.
⦿ Float the sections onto the surface of clean glass slides.
⦿ If the block is not ribboning well then place it back on the ice block to
cool off firm up the wax.
⦿ If the specimens fragment when placed on the water bath then it may be
too hot
⦿ Place the slides with paraffin sections in a 65°C oven for 20 minutes (so
the wax just starts to melt) to bond the tissue to the glass.
⦿ Slides can be stored overnight at room temperature
⦿ STEEL KNIVES
⦿ NON-CORROSIVE KNIVES FOR CRYOSTATS
⦿ DISPOSABLE BLADES
⦿ GLASS KNIVES
⦿ DIAMOND KNIVES
 STAINING

Purpose is to enhance natural contrast and to make various cells

and tissue components visible.
Routine Staining is Hematoxylin and Eosin staining
⦿The embedding process must be reversed
in order to get the paraffin wax out of the
tissue and allow water soluble dyes to
penetrate the sections.
⦿Therefore, before any staining can be
done, the slides are "deparaffinized" by
running them through xylen (or substitutes) to
alcohols to water.
⦿ There are no stains that ca n be done on
tissues containing paraffin.
⦿The staining process makes use of a
variety of dyes that have been chosen for
their ability to stain various cellular
components of tissue.
⦿ Theroutine stain is that of hematoxylin and eosin
(H and E).
⦿Other stains are referred to as "special stains"
because they are employed in specific situations
according to the diagnostic need.
H &E Staining Protocol
 Alcohol 70% ….2mint.
1) Removal of paraffin wax.
 Abs .Alcohol I  3mint
Xylene I  3mint
 Abs .Alcohol II 3mint
Xylene II  3mint
 Xylene I  3mint
2) Removal of xylol with abs .Alcohol.
 Xylene II 3mint.
Abs Alcohol I  3mint
Abs Alcohol II  3mint
3) Rx with descending grades of alcohol.
 Alcohol 70%  3mint
 Water  3mint  5mint.
 Hematoxylin 10-30 mints.
 Water 5mint -10mint.
 Decolorization acid alcohol 70% -2-5 dips.
 Water 3-5mints. Mordant: A metallic salt or hydroxide that
 Mordanting  Ammonia Alcohol 70% ….5mint. combines with a dye radical to form an insoluble
 Water  3-5mint. compound(take) permanent dye.
 Alcohol 70% , 3mint.
 Counter stain Eosin ½-2 mint
⦿ Slides being stained on automated Stainer
⦿ Frozen sections are stained by hand, because
this is faster for one or a few individual sections.

⦿ The stain is a "progressive" stain in which the section is left

in contact with the stain until the desired tint is achieved
⦿ Thestained section on the slide must be covered
with a thin piece plastic or glass to protect the tissues
from being scratched, to provide better optical quality for
viewing under the microscope and to preserve the tissue
section for years to come.

⦿ The stained slide must go through the reverse

process that it went through from
section to water.
⦿ The stained slide is taken through a ser

ha
permanent resinous substance beneath the glass
coverslip, a plastic film, can be placed over
or the
section.
Mounting of Cover Slip:

Put a drop of mounting medium (Canada balsam, DPX) which

has a similar refractive index to that of glass on the section.

 Place a glass cover slip over it.

 Avoid any air bubble above tissue section
 Allow it to dry in an incubator at 25-26C overnight.
Results of H & E Staining:
Nuclei Dark purple or blue –black.
All cytoplasmic structure  Red.
CartilagePink or light blue to dark blue.
Calcium and calcified bonepurplish blue.
Plasma cell osteoblast purplish.

RBC/keratin /eosinophil granule  bright red.

Collagen /osteoid tissue  light pink.
Muscle fiber/thyroid /thick elastic fiber/decalcified matrix  deep pink.