This document discusses tissue preparation techniques for immunohistochemistry. It begins by listing names and identification numbers. It then outlines the goals of being able to identify fixatives, understand sectioning, and describe whole mount preparation. It discusses various fixation methods and fixatives used including formalin, paraformaldehyde, glutaraldehyde, and acetone. It describes sectioning techniques using paraffin embedding and vibratome sectioning. It concludes by discussing advantages and disadvantages of different preparation methods and stresses that no single method is ideal for all antigens.

2. 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

3. By the end presentation, we able to:
 Identify the fixative used for immunohistochemistry.
 Understand the sectioning for immunohistochemistry.
 Described the whole mount preparation.

4. Fixation
Sectioning
Whole mount preparation

5.  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.

6.  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

7.  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).

8.  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.

9.  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.

10.  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.

11. A 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.

12. 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.

13.  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.

14. 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.

15.  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.

16.  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.

18. 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

19. 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.

20. 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.

21.  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“.

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