TISSUE PREPARATION

1. RAFEAH RUSLI 03-200904-00277

2. RAFIDAH ABRAHAM 03-200904-00324

3. SAMSON AK CLEMENT 03-200904-00359

4. SANDRA LOUIS 03-200904- 00274

5. SARTIKA AMRAN 03-200904-00180

6. VERA DIANE 03-200904-00244

By the end presentation, we able to:

Identify the fixative used for immunohistochemistry.

Understand the sectioning for immunohistochemistry.

Described the whole mount preparation.

OBJECTIVE

CONTENT

Fixation

Sectioning

Whole mount preparation

Tissue preparation is the cornerstone of immunohistochemistry.

To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential.

Inappropiate or prolonged fixation may significantly diminish the antibody binding capability.

There is no one universal fixative that is ideal for the demonstration of all antigens.

FIXATION

Many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections.

The discover and development of antigen retrieval techniques further enhanced the use of formalin as routine fixative for immunohistochemistry in many research laboratories. 

For best results, vertebrate tissues (especially neuronal tissues) usually require fixation by transcardial perfusion for optimal tissue preservation

The most common fixatives used for immunohistochemistry are the followings:- 4% paraformaldehyde in 0.1M phosphate buffer.- 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer.- PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M phosphate buffer.- 4% paraformaldehyde with 0.05% glutaraldehyde

(TEM immunohistochemistry). 

Some antigens will not survive even moderate amounts of aldehyde fixation.

Tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat without infiltrating with sucrose.

The sections should be kept frozen at -20 C or lower until fixation with cold acetone or alcohol.

The sections can be processed using standard immunohistochemical staining protocols.

Paraffin wax has remained the most widely used embedding

medium for diagnostic histopathology in routine histological

laboratories. The largest proportion of material for immunohistochemistry

is formalin-fixed, paraffin-embedded. Paraffin sections produce satisfactory results for the

demonstration of majority of tissue antigens with the use of

antigen retrieval techniques.

SECTIONING

Certain cell antigens do not survive routine fixation and

paraffin embedding. The disadvantage of frozen sections includes:

i) Poor morphology

Ii) Poor resolution at higher magnifications

Iii) Special storage needed

Iv) Limited retrospective studies and cutting difficulty over

paraffin sections.

VIBRATOMEA vibratome is an instrument that is similar

to a microtome but uses a vibrating razor

blade to cut through tissue.

The vibration amplitude, the speed, and

the angle of the blade can all be controlled.

Fixed or fresh tissue pieces are embedded

in low gelling temperature agarose.(Some

have had success without using the agarose

to embed.)

The resulting agarose block containing the

tissue piece is then glued to a metal block

and sectioned while submerged ina a water

or buffer bath.

Individual sections are then collected with

a fine brush and transferred to slides or

multiwell plates for staining.

Advantages of vibratome :

No need to dehydrate tissues prior to embedding, thus decreased loss of cell constituents.

No messy paraffin embedding. No need to deparaffinise and rehydrate sections prior to

immunostaining. No high temperatures or harsh chemical treatments that

may lead to antigen instability. No special microtome blades required. Less chance of artifacts caused by parrafin embedding or

freezing. Increased tissue autofluoresecence due to paraffin

embedding being avoided.

Less wait period from tissue sampling to time of immunolabelling.

The tissue is not processed through organic solvents or high

heat, which can destroy the antigenicity when

immunocytochemistry. The morphology of tissue sections is not disrupted due to no

freezing and thawing needed. Vibratome sections are often used for floating

immunostaining, especially for pre-embedding EM

immunohistochemistry.

The disadvantage of vibratome sections :

Instead of ribbons, single sections are cut and collected which are more delicate and difficult to handle.

Sections are generally thicker than those obtained with paraffin methods.

penetration of antibodies and other reagents may be slower and thus longer incubation times may be necessary.

thick sections may be difficult to image with the microscope. Securing vibratome sections to glass slides can be difficult or

impossible, due to the thickness of the sections. the sectioning process is slow and difficult with soft and

poorly fixed tissues. The chatter marks or vibratome lines are often appeared in

the sections.

Small blocks of tissue (less than 5 mm thick) can be processed as whole mounts.

The advantage of WMP :The results provide three dimensional information about the location of antigens without the need for reconstruction from sections.

Whole Mount Preparation

However, the major limitation of using whole mounts is antibody penetration may not be complete in the tissue, resulting in uneven staining or false negative staining.

Treatment : Triton X-100 or saponin are used routinely for whole mount immunohistochemistry to enhance penetration of the antibody.

MICROSCOPE OPTIC

Day 1: Dissection and Fixation of Mammary Glands 1. Dissect out mammary gland and spread on glass

slide. 2. Fix tissue O/N in Carnoy's fixative at RT.

Day 2: Rehydration and Staining of Glands 1. Wash in 70% EtOH 2 x 15 min. 2. Wash in 50% EtOH 2 x 10 min. 3. Wash in 30% EtOH 2 x 10 min. 4. Wash in 10% EtOH 2 x 10 min. 5. Wash in distilled H2O 1 x 5 min. 6. Stain O/N in carmine alum stain

Mammary Gland Whole Mount Preparations

Day 3: Clearing and Mounting of Glands 1. Wash in 70% EtOH 2 x 15 min. 2. Wash in 95% EtOH 2 x 15 min. 3. Wash in 100% EtOH 2 x 15 min. 4. Clear in xylene approximately 30 minutes

(or until fat is sufficiently cleared from glands).

5. Mount with - 1mL SecureMount and glass cover slip.

Carnoy's Fix: 6 parts 100% EtOH 3 parts CHCl3 1 part glacial acetic acid

Carmine Alum Stain: Place 1g carmine (Sigma C1022) and 2.5g

aluminum potassium sulfate (Sigma A7167) in 500mL dH20 and boil 20 min. Adjust final vol. to 500mL with H20. Filter and add thymol crystal as preservative. Refrigerate. Can be used for several months. Discard when color becomes weak.

There is no standard tissue preparation schedule for the optimal demonstration of all antigens.

Factors involved in all aspects of tissue preparation can affect immunoreactivity, so it is important that precise details of the preparation schedule are given when reporting immunocytochemical studies, rather than using the general term "routinely fixed and processed“.

CONCLUSION

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