1 Overview

1.1 Purpose and Scope

This protocol establishes a routine operating procedure for muscle fiber staining using the Puchtler tannic acid–azophloxine method. It is suitable for differentiating muscle fibers and collagen fibers in paraffin-embedded tissue sections. It can be used for morphological studies of smooth muscle, skeletal muscle, and cardiac muscle tissues, and may also assist in displaying myoepithelial cell components in tissues such as breast and skin.

1.2 Method Principle

(1) This method is a sequential differential staining system. Through hematoxylin nuclear staining, tannic acid differentiation, phosphomolybdic acid treatment, and azophloxine counterstaining, different tissue components acquire stable color differences.

(2) Lea hematoxylin is mainly used for nuclear staining, turning cell nuclei blue and facilitating subsequent tissue structure recognition.

(3) After tannic acid and phosphomolybdic acid treatment, collagen fibers and muscle fibers show different binding capacities for azophloxine. As a result, muscle fibers and myoepithelial cells appear rose-red, while collagen fibers appear yellow.

1.3 Staining Quality Requirements

(1) Muscle fibers and myoepithelial cells should be clearly stained and show a stable rose-red color.

(2) Collagen fibers should be clearly differentiated and appear yellow.

(3) Cell nuclei should be clearly blue-stained, the background should be relatively clean, and the tissue outline should remain intact.

2 Materials and Reagents

2.1 Sample Types

(1) Paraffin sections of muscle-related tissues.

(2) Paraffin sections of breast tissue.

(3) Paraffin sections of skin tissue.

(4) Other routine tissue sections requiring differentiation between muscle fibers and collagen fibers.

2.2 Main Reagents

(1) Bouin’s fixative, Carnoy’s fixative, or neutral formalin.

(2) 95% ethanol.

(3) Absolute ethanol.

(4) Lea hematoxylin staining solution.

(5) Tannic acid differentiation solution.

(6) Phosphomolybdic acid differentiation solution.

(7) Azophloxine staining solution.

(8) Acid alcohol differentiation solution.

(9) Distilled water.

(10) Xylene or deparaffinization/clearing solution.

(11) Neutral balsam.

2.3 Consumables and Equipment

(1) Glass slides and coverslips.

(2) Staining jars.

(3) Forceps and absorbent paper.

(4) Paraffin microtome.

(5) Slide baking equipment.

(6) Microscope.

3 Staining Solution and Sample Preparation

3.1 Fixation Requirements

(1) Routine tissues are preferably fixed in Bouin’s fixative for 2–3 h, which generally helps obtain better staining contrast.

(2) Carnoy’s fixative or neutral formalin may also be used, but some fixation systems may result in weaker staining.

(3) Samples in the same batch should use the same fixation system and fixation time whenever possible to reduce staining variation.

3.2 Dehydration, Embedding, and Sectioning Requirements

(1) After fixation, samples may be directly transferred into 95% ethanol and then dehydrated and embedded according to routine procedures.

(2) The recommended thickness of paraffin sections is 4 μm.

(3) Section thickness should be kept as consistent as possible to improve staining uniformity and comparability within the same batch.

3.3 Deparaffinization Requirements

(1) Paraffin sections should be routinely deparaffinized with xylene or deparaffinization/clearing solution and brought to water.

(2) Incomplete deparaffinization may lead to uneven staining or gray background.

(3) Before entering the staining steps, sections should be fully hydrated.

4 Experimental Procedure

4.1 Fixation and Embedding

(1) Fix routine tissue in Bouin’s fixative for 2–3 h.

(2) After fixation, transfer directly into 95% ethanol.

(3) Complete dehydration and embedding according to routine procedures.

4.2 Sectioning and Deparaffinization

(1) Prepare paraffin sections with a thickness of 4 μm.

(2) Routinely deparaffinize with xylene or deparaffinization/clearing solution and bring to water.

4.3 Nuclear Staining

(1) Immerse the sections in Lea hematoxylin staining solution for 8–10 min.

(2) Remove and rinse under running water for 10 min.

(3) Nuclear staining should be sufficient to produce clearly blue-stained nuclei and should not be too weak.

4.4 Tannic Acid Differentiation

(1) Place the sections in tannic acid differentiation solution for 10 min.

(2) Rinse under running water for 1 min after treatment.

(3) This step helps achieve differential coloration between collagen fibers and muscle fibers in the subsequent steps.

4.5 Phosphomolybdic Acid Differentiation

(1) Place the sections in phosphomolybdic acid differentiation solution for 10 min.

(2) After treatment, proceed directly to the azophloxine staining step.

(3) The duration of this step should be kept consistent to reduce batch-to-batch variation in color differentiation.

4.6 Azophloxine Staining

(1) Immerse the sections in azophloxine staining solution for 10–15 min.

(2) The staining time may be adjusted slightly according to tissue type and staining intensity.

(3) Muscle fibers should be sufficiently stained without overstaining.

4.7 Acid Alcohol Differentiation

(1) Place the sections in acid alcohol differentiation solution for 3–5 s.

(2) Differentiation should be controlled until excess red dye is mostly removed from the section and the muscle fibers appear bright red or rose-red.

(3) This is one of the key control points of the method. Insufficient or excessive differentiation should be avoided.

4.8 Dehydration, Clearing, and Mounting

(1) After staining, dehydrate repeatedly with absolute ethanol and complete dehydration through a graded ethanol series.

(2) Transfer into xylene or deparaffinization/clearing solution for clearing.

(3) Add neutral balsam, cover with a coverslip, and complete mounting.

(4) Avoid air bubbles after mounting and ensure even distribution of the mounting medium.

5 Staining Results

5.1 Muscle Fibers and Myoepithelial Cells

(1) Muscle fibers appear rose-red.

(2) Myoepithelial cells appear rose-red.

5.2 Collagen Fibers

(1) Collagen fibers appear yellow.

(2) There should be clear color contrast between collagen fibers and muscle fibers.

5.3 Cell Nuclei

(1) Cell nuclei appear blue.

(2) Nuclear staining should clearly show tissue outlines and cellular localization relationships.

6 Key Points for Result Interpretation

6.1 Interpretation of Muscle Fibers

(1) Clear and continuous staining of muscle fibers indicates good preservation of muscle tissue structure.

(2) If muscle fiber staining is weak, first review the fixation system, azophloxine staining time, and acid alcohol differentiation degree.

6.2 Interpretation of Collagen Fibers

(1) Clear yellow distribution of collagen fibers helps observe the boundary between stromal components and muscle tissue.

(2) If collagen fibers and muscle fibers are not clearly separated, first check the tannic acid differentiation and phosphomolybdic acid differentiation steps.

6.3 Interpretation of Myoepithelial Cells

(1) Rose-red staining of myoepithelial cells helps auxiliary identification in tissues such as breast and skin.

(2) Interpretation should be performed together with nuclear position and tissue structural background.

7 Common Problems and Cause Analysis

7.1 Weak Muscle Fiber Staining

(1) The fixation system is unsuitable, especially when Bouin’s fixative is not used.

(2) Azophloxine staining time is insufficient.

(3) Acid alcohol differentiation is excessive.

7.2 Overall Red Appearance of the Section

(1) Acid alcohol differentiation is insufficient.

(2) Azophloxine staining is too strong.

(3) Excess dye is not effectively removed before dehydration.

7.3 Poor Differentiation Between Muscle Fibers and Collagen Fibers

(1) Tannic acid differentiation is insufficient.

(2) Phosphomolybdic acid differentiation time is insufficient or inconsistent.

(3) Differences in tissue fixation and section thickness cause batch-to-batch variation.

7.4 Unclear Nuclear Staining

(1) Lea hematoxylin staining time is insufficient.

(2) Running water rinsing is insufficient or excessive.

(3) Incomplete deparaffinization affects nuclear staining uniformity.

8 Application Scope

8.1 Muscle Tissue Observation

(1) Observation of smooth muscle-related tissues.

(2) Observation of skeletal muscle and cardiac muscle-related tissues.

8.2 Connective Tissue Differentiation

(1) Used to distinguish muscle fibers from collagen fibers.

(2) Suitable for observing muscle layer continuity and stromal collagen distribution.

8.3 Myoepithelial-Related Research

(1) Can display myoepithelial cells.

(2) Applicable to morphological studies of breast, skin, and other tissues containing myoepithelial cell components.

9 Safety and Operating Guidelines

9.1 Personal Protection

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

(2) Handle fixatives, acid alcohol, xylene, and staining solutions in a well-ventilated environment.

9.2 Waste Disposal

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

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

10 Selection of Related Reagents and Materials

Table 1 Reagent Selection for Puchtler Tannic Acid–Azophloxine Muscle Fiber Staining

For more related articles, please see below:

[1] Staining-Based Differentiation of Collagen Fibers, Muscle Fibers, and Fibrin in Tissue Sections