Protocol of Histopathology Techniques for Tissue Processing to Slide Staining

Introduction

Histopathology means the diagnosis/ study of diseased/abnormal tissue/a cell under a microscope. It is performed by examining a thin tissue section on a glass slide under the microscope. In this histo-technique, a number of procedures are required to make the slide visualize under the microscope.

Histopathological technique/ processing steps for specimen /tissue are:

1-Collection

2-Fixation

3- Grossing

4- Processing

5- Embedding

6- Sectioning

7-Staining

Material Collection and Fixation

§ Sample container according to the size of the specimen

§ 10% Neutral buffered formalin

Sodium phosphate, monobasic 4.0 gm

Sodium phosphate, dibasic 6.5 gm

Formaldehyde, 37% 100.0 ml

Distilled water 900.0 ml

SAFETY: Work in a well-ventilated area, wear goggles, gloves, and lab coat. Call Safety Services for large spills, and use a formaldehyde spill kit for small spills of 1000 ml. Formaldehyde: severe eye and skin irritant. Sensitizer by the skin and respiratory contact. Toxic by ingestion and inhalation. Target organ effects on the respiratory system.

Corrosive. Carcinogen.

Fixation (prevents specimen /tissue from autolysis and putrefaction)

Fix the specimen in 10% NBF for 4 hours to 72 hours according to the sample size.

Grossing

The term "grossing" means examining, describing, measuring, and sectioning the tissue (3-4mm thickness, if required) to be processed for diagnosis.

Processing steps

Tissue Processing time in Automatic tissue processor

Reagent Name	Time
Formalin -1	1 hour
Formalin-2	1 hour
Formalin-3	1 hour
Alcohol-1-60%	1 hour
Alcohol-2-70%	1 hour
Alcohol-3-80%	1 hour
Alcohol-4-90%	1 hour
Alcohol-5-100%	1 hour
Xylene-1	1 hour
Xylene-2	1 hour
Paraffin wax-1	1 hour
Paraffin wax-2	1 hour

Automatic Tissue Processor Unit

Embedding

This s the technique in which tissue/cells are placed in a supporting medium called paraffin wax to make the block, so that thin sections can be prepared by using a microtome.

Sectioning

Microtome is an instrument used for Sectioning the paraffin-embedded tissue at 3-5 microns to make slides from tissue

Staining

The slides prepared from the block are colorless and are not read under a microscope. Staining them with different colored dyes, makes the slides identify and study their morphology

Hematoxylin and Eosin (H&E) is the most frequently used stain in histology.

Preparation of Harris’s hematoxylin is as follows:

A- Hematoxylin -5gm in Absolute alcohol 50ml

B-Ammonium alum -100gm in Distilled water 1000ml

Mercuric oxide 2.5gm

Glacial acetic acid 4-5ml

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