1 Overview

1.1 Purpose and Scope of Application

This protocol is used to establish a routine staining procedure for the Thomas phosphomolybdic acid hematoxylin method. It is suitable for displaying collagen fibers, coarse reticular fibers, and argyrophilic cell granules in paraffin-embedded tissue sections. It can be used for observing connective tissue components, identifying coarse reticular structures, and conducting morphological studies of certain tissues containing argyrophilic granule-bearing cells.

1.2 Principle

(1) The Thomas phosphomolybdic acid hematoxylin method is a phosphomolybdic acid-mordanted hematoxylin staining method. It mainly achieves selective coloration through differences in the affinity of the phosphomolybdic acid-hematoxylin system for different tissue components.

(2) This method provides good visualization of collagen fibers, coarse reticular fibers, and argyrophilic cell granules. Collagen fibers and coarse reticular fibers usually appear purple to black, while argyrophilic cell granules appear black.

(3) Cell nuclei are usually pale blue in this staining system, helping create layered contrast with deeply stained fibers and granular structures.

1.3 Staining Quality Requirements

(1) Collagen fibers and coarse reticular fibers should be clearly stained, appearing purple to black.

(2) Argyrophilic cell granules should be clearly stained black.

(3) Cell nuclei should be pale blue, the background should be relatively clean, and tissue contours should remain intact.

2 Materials and Reagents

2.1 Sample Types

(1) Routine paraffin-embedded tissue sections.

(2) Tissue sections requiring visualization of collagen fibers or coarse reticular fibers.

(3) Related tissue sections containing argyrophilic cell granules.

2.2 Main Reagents

(1) Thomas phosphomolybdic acid hematoxylin staining solution.

(2) Thomas differentiation solution.

(3) Graded ethanol.

(4) Distilled water.

(5) Xylene or deparaffinization clearing solution.

(6) Neutral balsam.

2.3 Self-Prepared Fixation System

(1) Routine histological fixatives.

(2) Avoid dichromate-based fixatives.

2.4 Consumables and Equipment

(1) Glass slides and coverslips.

(2) Staining jars.

(3) Forceps and absorbent paper.

(4) Paraffin microtome.

(5) Microscope.

3 Staining Solution and Sample Preparation

3.1 Fixation Requirements

(1) Tissue samples should be fixed, dehydrated, and embedded according to routine methods.

(2) Dichromate-based fixatives are not recommended, as they may affect staining performance.

(3) Samples within the same batch should preferably use consistent fixation and embedding conditions to reduce batch-to-batch variation.

3.2 Sectioning Requirements

(1) Paraffin section thickness should be kept consistent.

(2) Sections should adhere firmly to the slides to prevent detachment during deparaffinization and differentiation.

(3) Before staining, sections should be fully deparaffinized and hydrated to water.

3.3 Deparaffinization and Hydration Requirements

(1) Paraffin sections should be deparaffinized with xylene or deparaffinization clearing solution.

(2) Sections should then be gradually hydrated to water through graded ethanol.

(3) Incomplete deparaffinization or insufficient hydration may result in uneven staining.

4 Experimental Procedure

4.1 Deparaffinization and Hydration

(1) Prepare paraffin sections after routine tissue fixation, dehydration, and embedding.

(2) Deparaffinize sections with xylene or deparaffinization clearing solution.

(3) Hydrate sections to water through graded ethanol.

4.2 Thomas Phosphomolybdic Acid Hematoxylin Staining

(1) Place sections in Thomas phosphomolybdic acid hematoxylin staining solution and stain for 2 min.

(2) Remove the sections and rinse with distilled water.

(3) The staining time should not be too long, to avoid deepening of the background.

4.3 Differentiation with Thomas Differentiation Solution

(1) Add Thomas differentiation solution dropwise onto the sections for differentiation.

(2) Immediately rinse quickly with distilled water after differentiation.

(3) Then rinse with tap water for 2 min.

(4) This step should be controlled until the background becomes lighter while the target structures remain clearly and deeply stained.

4.4 Dehydration, Clearing, and Mounting

(1) Rapidly dehydrate sections with 95% ethanol and absolute ethanol.

(2) Continue dehydration according to the routine procedure.

(3) Transfer sections into xylene or deparaffinization clearing solution for clearing.

(4) Add neutral balsam, apply the coverslip, and complete mounting.

(5) After mounting, avoid air bubbles and keep the mounting medium evenly distributed.

5 Staining Results

5.1 Collagen Fibers and Coarse Reticular Fibers

(1) Collagen fibers appear purple to black.

(2) Coarse reticular fibers appear purple to black.

5.2 Argyrophilic Cell Granules

(1) Argyrophilic cell granules appear black.

5.3 Cell Nuclei

(1) Cell nuclei appear pale blue.

6 Key Points for Result Interpretation

6.1 Interpretation of Fiber Components

(1) Clearly and deeply stained collagen fibers and coarse reticular fibers facilitate observation of stromal structures and fiber distribution.

(2) If the target fibers are weakly stained, first check the staining time, degree of differentiation, and fixation conditions.

6.2 Interpretation of Argyrophilic Cell Granules

(1) When argyrophilic cell granules appear black, they are usually easy to distinguish from surrounding tissues.

(2) Interpretation should be integrated with the distribution of granules and the tissue background.

6.3 Interpretation of Cell Nuclei

(1) Pale-blue nuclei can serve as references for tissue localization and structural orientation.

(2) If nuclear staining is too dark, observation of deeply stained fibers and granules may be affected.

7 Common Problems and Cause Analysis

7.1 Target Structures Are Weakly Stained

(1) Staining time is insufficient.

(2) Differentiation is excessive.

(3) The fixation system is unsuitable; dichromate-based fixatives are especially likely to affect staining.

7.2 Background Is Too Dark

(1) Thomas phosphomolybdic acid hematoxylin staining time is too long.

(2) Differentiation is insufficient.

(3) Sections are incompletely deparaffinized or contain excessive tissue residues.

7.3 Target Structures and Background Are Poorly Differentiated

(1) Differentiation solution treatment is unstable.

(2) Rinsing is not performed promptly, resulting in inconsistent differentiation endpoints.

(3) Inconsistent section thickness or fixation conditions within the same batch causes staining variation.

7.4 Sections Appear Gray or Have Poor Transparency

(1) Dehydration is insufficient.

(2) Clearing is insufficient.

(3) Residual water remains before mounting, affecting microscopic observation.

8 Application Scope

8.1 Connective Tissue Observation

(1) Used to display collagen fiber distribution.

(2) Used to display coarse reticular fiber distribution.

8.2 Observation of Argyrophilic Cell-Related Structures

(1) Used to observe argyrophilic cell granules.

(2) Suitable for auxiliary identification of related cellular components.

8.3 Histological Morphology Research

(1) Suitable for contrast visualization of specific fiber components in routine tissue sections.

(2) Can serve as a supplementary observation method alongside other fiber staining methods.

9 Safety and Operating Requirements

9.1 Personal Protection

(1) Wear a laboratory coat and disposable gloves during the experiment.

(2) Xylene, deparaffinization solution, and staining solutions should be handled in a well-ventilated environment.

9.2 Waste Disposal

(1) Waste liquids containing ethanol, xylene, and residual staining solutions should be collected separately.

(2) All waste liquids and contaminated consumables should be disposed of according to laboratory chemical waste handling regulations.

10 Reagent Selection for the Thomas Phosphomolybdic Acid Hematoxylin Method