1 Overview

1.1 Purpose and Scope of Application

This protocol is used to establish a routine operating procedure for muscle fiber staining by the Puchtler tannic acid–azo phloxine method. It is suitable for differential observation of muscle fibers and collagen fibers in paraffin-embedded tissue sections. It can be used for morphological studies of tissues related to smooth muscle, skeletal muscle, and cardiac muscle, and can also assist in displaying tissues containing myoepithelial cell components, such as breast and skin tissues.

1.2 Principle

(1) The tannic acid–azo phloxine method is a differential staining method. Its core principle is that two acidic dyes act sequentially and produce differential coloration in different tissue components.

(2) Hematoxylin staining solution is first used for nuclear staining, turning nuclei blue and facilitating subsequent recognition of tissue structures.

(3) Tannic acid more readily penetrates collagen fiber systems with relatively high permeability. After subsequent processing, collagen fibers appear yellow.

(4) Azo phloxine more readily penetrates muscle fiber systems with relatively lower permeability, staining muscle fibers and myoepithelial cells rose-red. This enables clear differentiation from collagen fibers.

1.3 Staining Quality Requirements

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

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

(3) Nuclei should be clearly stained blue, with a clean background and intact tissue contours.

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) Hematoxylin staining solution.

(2) Tannic acid differentiation solution.

(3) Phosphomolybdic acid differentiation solution.

(4) Azo phloxine staining solution.

(5) Acid alcohol differentiation solution.

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

(7) 95% ethanol.

(8) Absolute ethanol.

(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 tissue is preferably fixed in Bouin’s fixative for 2–3 h, which usually helps obtain better staining contrast.

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

(3) Samples within the same batch should preferably use a consistent fixation system and fixation time 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 paraffin section thickness 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 a grayish 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, directly transfer the tissue into 95% ethanol.

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

4.2 Sectioning and Deparaffinization

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

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

4.3 Nuclear Staining

(1) Immerse sections in Hematoxylin staining solution for 8–10 min.

(2) Remove the sections and rinse in running water for 10 min.

(3) Nuclear staining should produce clear blue nuclei and should not be too weak.

4.4 Tannic Acid Differentiation

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

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

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

4.5 Phosphomolybdic Acid Differentiation

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

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

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

4.6 Azo Phloxine Staining

(1) Immerse sections in azo phloxine staining solution for 10–15 min.

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

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

4.7 Acid Alcohol Differentiation

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

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

(3) This step is one of the key control points of this method. Under-differentiation or over-differentiation should be avoided.

4.8 Dehydration, Clearing, and Mounting

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

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

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

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

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 Nuclei

(1) Nuclei appear blue.

(2) Nuclear staining should clearly show tissue contours and the positional relationship of cells.

6 Key Points for Result Interpretation

6.1 Interpretation of Muscle Fibers

(1) Bright 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, azo phloxine staining time, and degree of acid alcohol differentiation.

6.2 Interpretation of Collagen Fibers

(1) Clearly distributed yellow collagen fibers facilitate observation of the boundary between stromal components and muscle tissue.

(2) If the boundary between collagen fibers and muscle fibers is unclear, first check the tannic acid differentiation and phosphomolybdic acid differentiation steps.

6.3 Interpretation of Myoepithelial Cells

(1) Rose-red myoepithelial cells can assist identification in tissues such as breast and skin.

(2) Interpretation should be integrated with nuclear position and the surrounding tissue structure.

7 Common Problems and Cause Analysis

7.1 Weak Muscle Fiber Staining

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

(2) Azo phloxine staining time is insufficient.

(3) Acid alcohol differentiation is excessive.

7.2 Entire Section Appears Too Red

(1) Acid alcohol differentiation is insufficient.

(2) Azo phloxine staining is too intense.

(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 unstable.

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

7.4 Unclear Nuclear Staining

(1) Hematoxylin staining time is insufficient.

(2) Running water rinsing is insufficient or excessive.

(3) Incomplete deparaffinization affects the uniformity of nuclear staining.

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 Cell-Related Research

(1) Can display myoepithelial cells.

(2) Suitable for morphological studies of tissues containing myoepithelial cell components, such as breast and skin.

9 Safety and Operating Requirements

9.1 Personal Protection

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

(2) Fixatives, acid alcohol, xylene, and staining solutions should be handled in a well-ventilated environment.

9.2 Waste Disposal

(1) Waste liquids containing fixatives, 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 Muscle Fiber Staining (Puchtler Tannic Acid–Azo Phloxine Method)

For more related articles, please see below:

[1] Experiments on the resolution of electromyographic signals in muscle fibers

[2] Isolation and culture of single muscle fibers from skeletal muscle