Neuromuscular junction visualization in paraffin‐embedded thyroarytenoid muscle sections: Expanding options beyond frozen section analysis

Abstract

Objective(s)

The current gold standard for immunofluorescent (IF) visualization of neuromuscular junctions (NMJs) in muscle utilizes frozen tissue sections with fluorescent conjugated antibodies to demarcate neurons and IF alpha‐bungarotoxin (α‐BTX) to demarcate motor endplates. Frozen tissue sectioning comes with inherent inescapable limitations, including cryosectioning artifact and limited sample shelf‐life. However, a parallel approach to identify NMJs in paraffin‐embedded tissue sections has not been previously described.

Methods

Yucatan minipig thyroarytenoid (TA) muscle was harvested and prepared as 5‐μm thick paraffin‐embedded tissue sections. A variety of antibodies at various concentrations were selected to target nicotinic acetylcholine receptors, synaptic vesicles, and neurons.

Results

Neurofilament (NEFL, Invitrogen, 1:500) and synaptic vesicle glycoprotein (SV2, DSHB, 1:230) bound and demarcated the neurons and synaptic vesicles, respectively. Following consistent visualization of nerve tissue, rabbit anti‐nicotinic acetylcholine receptor alpha‐1 subunit (CHRNα₁, Abcam, 1:500) was used to identify the acetylcholine receptors within motor endplates. Complete NMJ visualization was then achieved with an optimized protocol using primary antibodies to the neurofilament light chain, nerve synaptic vesicle glycoprotein 2, and the alpha 1 subunit of the nicotinic acetylcholine receptor. Slide imaging was performed with the Echo Revolve Microscope (40×).

Conclusions

Herein, we describe a new methodology to visualize NMJs within paraffin‐embedded TA muscle sections. Our protocol avoids the known limitations associated with cryosectioned samples and introduces a new neurolaryngologic research tool that utilizes the advantageous ability of paraffin‐embedded sectioning to preserve tissue morphology. In conjunction with standard cryosectioned methods, the described paraffin‐embedded protocol serves to enhance histological analysis of NMJs.

Level of evidence

NA.

Herein, we describe a new methodology to visualize neuromuscular junctions (NMJs) within formalin fixed paraffin‐embedded (FFPE) thyroaryteniod (TA) muscle sections. Our protocol avoids the known limitations associated with cryosectioned samples and should be widely applicable for histologic analysis in animal and human studies.

1. INTRODUCTION

In all fields of medicine that focus on nerve injury and regeneration, effective study of nerve‐to‐muscle contact is vital. ¹ Neurolaryngologic studies focusing on interventions for vocal fold paralysis are no exception, in which examination of muscle reinnervation lays at the core of analysis. ² The standard method for histological analysis of neuromuscular junctions (NMJs) has changed over the past few decades. Notable staining protocols in the past include cholinesterase and silver‐gold staining methods. ³ , ⁴ The current, widely accepted, ‘gold’ standard for NMJ staining includes the utilization of alpha‐bungarotoxin (α‐BTX) due to its selective reaction with nicotinic acetylcholine receptors (CHRNs) that reside at motor endplates (coupled with anti‐neurofilament and anti‐synaptic vesicle stains). ⁵ , ⁶ NMJ staining protocols provide essential data about the innervation status of a given muscle, with many experiments quantifying laryngeal muscle innervation based on the percentage of motor endplates with neuronal contact. ⁴ , ⁵ Although staining protocols have improved throughout time, preservation of tissue samples for visualization has improved only marginally given the reliance on frozen tissue processing in all of the aforementioned protocols. In addition to alteration to tissue morphology (due to direct manipulation and cryosectioning (freeze) artifact), the current standard methods are also limited by the need for storage at −80°C and shelf life of only 1 year. ⁷ Thus, room for further improvement still exists with respect to NMJ histological analysis.

Formalin fixed paraffin‐embedded (FFPE) sectioning offers a route toward effective histological visualization through improved tissue morphology preservation, longer shelf life (multiple years), and the ability for storage at room temperature. ⁷ FFPE processed slides have yet to be incorporated into mainstream neurolaryngologic research analysis owing to the fact that α‐BTX cannot be utilized in this approach. Specifically speaking, the utility of α‐BTX relies on a chemical reaction rather than antibody binding. The paraffin‐based (wax) processing blocks the chemical reaction between CHRN within NMJs and α‐BTX. For a reliable FFPE method for NMJ visualization to be developed, an alternative to α‐BTX is needed.

Herein, we describe a methodology to visualize NMJs within paraffin‐embedded thyroarytenoid (TA) muscle sections that was developed by studying the inherent structural properties of NMJs, that minimizes limitations of current standard frozen sectioning including limited sample shelf life, tissue manipulation artifact, and cryosectioning artifact. To our knowledge, to date no alternative NMJ staining protocol for paraffin‐embedded samples has been described in the literature.

2. METHODS

A graphical illustration of the study is illustrated in Figure 1.

FIGURE 1.

Overview of staining approach. Literature review of neuromuscular junction structure helped guide prospective antibody targets for staining which further guided identification of suitable antibodies from manufacturers. Full Yucatan minipig larynges were harvested and transected at midline (denoted by line) to allow for full visualization of TA muscles. Harvested TA muscle samples were processed via formalin fixed paraffin‐embedded (FFPE) method into 5‐μm thick sections. Following rehydration utilizing an ethanol‐deionized water gradient, samples were stained with selected antibodies. Stained slides were then efficiently visualized on an Echo Revolve Microscope at 40× on fluorescent settings. Figure created in part with Biorender.com.

2.1. Animal model, tissue harvest, and tissue processing

This animal study protocol was approved by the Purdue Institutional Animal Care and Use Committee (Purdue IACUC). Institutional guidelines, in accordance with the National Institutes of Health (NIH), were followed for the handling and care of animals. Yucatan minipigs (S&S Farms, Malta, IL) from a separate study were humanely euthanized although still under anesthesia according to NIH and Purdue IACUC approved methods. Full larynges were excised and dedicated for this histological study. Larynges were placed in 1× Phosphate Buffered Saline (PBS, ThermoFisher Scientific, Franklin, MA). Dissected TA muscles were fixed in 4% formalin (ThermoFisher), and subsequently paraffin‐embedded and serial sectioned at 5‐μm thickness.

2.2. Staining approach

Successful selection of antibody targets relied on reviewing the structure of NMJs. Slides were prepared and stained in a similar, standardized fashion previously described by our lab. ⁸ Following identification of appropriate antibodies, specimens were deparaffinized, rehydrated, and underwent a heat‐based citrate antigen retrieval (Vector Laboratories, Newark, CA). Slides were blocked in 4% Fetal Bovine Serum (HyClone, Logan, UT), 2% Triton X‐100 (ThermoFisher) in 1× PBS for 60 min at room temperature, were permeabilized with 4% Triton X‐100 for 15 min, and subsequently were incubated with TrueBlack Lipofuscin Autofluorescence Quencher (Biotium, Fremont, CA) for 45 s. Slides were then incubated in a cocktail of all primary antibodies in the aforementioned blocking serum, in total darkness at 4°C, overnight. The next day, specimens were further incubated in a cocktail of all secondary antibodies in the aforementioned blocking serum, in total darkness at room temperature, for 1 h. DAPI Fluoromount‐G (SouthernBiotech, Birmingham, AL) was added, a coverslip was affixed, and slides were set to dry in total darkness, at room temperature, overnight. An Echo Revolve microscope (Echo, San Diego, CA) was utilized to visualize the specimens.

3. RESULTS

3.1. Selection of antibodies targets

Review of NMJ structure was integral in validating the staining protocol. Figure 2 highlights key structural components of an NMJ and specifically the pentameric transmembrane nicotinic acetylcholine receptor at the end of muscle cells. ⁹

FIGURE 2.

Diagrams highlighting key structural components of a NMJ (A) and a nicotinic acetylcholine receptor (B). Key to visualizing nerve to muscle contact includes proper targeting of nerve fibers via neurofilament light chain (NEFL), nerve terminals via synaptic vesicle glycoprotein (SV2), and multiple nicotinic acetylcholine receptors on muscle cell surfaces (CHRN). The nicotinic acetylcholine receptor on skeletal muscle (CHRN) is a pentameric transmembrane receptor. Stars (*) denote targets identified for antibody staining. Figure created with Biorender.com.

Accordingly, anti‐neurofilament light chain, anti‐synaptic vesicle glycoprotein (SV2), and anti‐nicotinic acetylcholine receptors (CHRN, alpha‐1 subunit) antibodies were utilized to target nerve fibers, nerve terminals, and muscle cell surfaces, respectively. ¹⁰ Our finalized FFPE protocol for staining NMJs is outlined in Table 1. Primary antibodies include anti‐NEFL (ab2532995, 1:500, Invitrogen, Waltham, MA), anti‐SV2 (ab2315387, 1:230, DSHB, Iowa City, IA), and anti‐CHRNα₁ (ab135272, 1:500, Abcam, Waltham, MA) antibodies. Secondary antibodies include IgG H&L TRITC (ab6817, 1:200, Abcam) and IgG H&L Cy5 (ab2534032, 1:200, Invitrogen). Furthermore, to confirm distinct staining of NEFL and SV2, we stained additional slides utilizing a rabbit‐derived neurofilament stain (ab9568, 1:500, Abcam).

TABLE 1.

Specific antibodies in finalized protocol for visualizing NMJs in minipig TA muscle.

NMJ target	1° antibody	Concentration	2° antibody
NEFL	ab2532995 (Invitrogen, mouse‐derived) a	1:500	Donkey anti‐mouse TRITC
SV2	ab2315387 (DSHB, mouse‐derived) b	1:230	Donkey anti‐mouse TRITC
CHRNα1	ab135272 (Abcam, rabbit‐derived) c	1:500	Goat anti‐rabbit Cy5

Note: Specific antibody number, supplier, and concentration diluted in blocking serum (4% fetal bovine serum, 2% Triton X‐100 in phosphate buffered saline).

^a ThermoFisher Scientific Invitrogen. ¹³

^b Developmental Studies Hybridoma Bank. ¹⁴

^c Abcam. ¹⁵

3.2. Immunofluorescent visualization of NMJs in TA muscles

Nerve to muscle contact was effectively and consistently visualized on an Echo Revolve Microscope at 40× on the fluorescent settings. Figure 3 demonstrates immunofluorescently labeled NMJs of TA muscle within paraffin‐embedded section as visualized on fluorescent microscopy, with channels split to highlight different antibodies successfully utilized in our protocol. Distinct staining of NEFL and SV2 was also confirmed when using a rabbit‐derived neurofilament stain as seen in Figure S1.

FIGURE 3.

Immunofluorescently labeled NMJs of TA muscle within paraffin‐embedded section as visualized on fluorescent microscopy. Respective fluorescent channels are split to highlight the antibodies utilized in our protocol. The TRITC channel highlights visualization of nerve fibers (NEFL, ab2532995) and synaptic vesicle glycoprotein (SV2, ab2315387). The Cy5 channel displays visualization of nicotinic acetylcholine receptors on muscle cells (CHRNα₁, ab135272). The counterstain DAPI channel allows for visualization of skeletal muscle cell nuclei. Upon visualizing all the channels merged together, one can effectively differentiate between individual nerve fascicles between muscle cells (e.g. denoted by red arrow, as seen by no overlapping Cy5 staining), and sites of nerve‐to‐muscle contact at NMJs (e.g. denoted by green arrow) (microscope = echo revolve, zoom = 40×, scale bar = 50 μm, red = SV2 and NEFL, green = CHRNα₁, blue = nuclei).

4. DISCUSSION

In this study, we examined a novel immunofluorescent staining protocol for visualizing NMJs in porcine TA muscles. As seen in Figure 3, the current protocol allows visualization of NMJs in paraffin‐embedded muscle sections with exceptionally preserved tissue morphology, notably allowing slides to be free of shearing and frozen artifacts. Additionally, the findings of this study provide greater flexibility to researchers with a greater affordance in time between tissue processing and eventual analysis.

The protocol can be widely applied to neurolaryngologic investigations that quantify the percentage of motor endplates with neuronal contact as an outcome for laryngeal muscle innervation status. ⁴ , ⁵ , ⁸ , ¹¹ In fact, the preserved nature of the tissue in the paraffin‐embedded sections may allow such quantification approaches to be more accurate in reflecting underlying innervation status. Overall, our study supports the notion for the inclusion of formalin fixed paraffin‐embedded protocols within histological analysis, thereby expanding options researchers have at their disposal when optimizing study results. ¹²

4.1. Additional applicability

Of note, although this protocol was developed for paraffin‐embedded muscle, all antibodies can be readily adaptable for frozen tissue samples. Additionally, per the antibody manufacturers all antibodies are compatible with mouse and human samples. ¹³ , ¹⁴ , ¹⁵

4.2. Limitations

Due to these sections being sliced at a thin 5 μm depth, full visualization of nerve from fascicle to muscle contact was challenging. However, as seen in Figure 3, the ability to differentiate between nerve fascicle and nerve contacting muscle as well as the proximity of free‐standing nerve fibers to NMJs, allows for sufficient visualization. Additionally, this limitation can likely be addressed by cutting thicker sections. Furthermore, although paraffin‐embedded sections allow for the avoidance of the limitations of frozen embedded sections, they are not without their own drawbacks. Although not applicable to this study, longer processing time and reduced ability for nucleic acid extraction are a few notable limitations. ¹⁶

Although the described protocol can be widely incorporated into the analysis of NMJ morphology, it remains to be seen if this paraffin‐embedded methodology can fully replace frozen section analysis. The purpose of this manuscript was to briefly communicate a novel histology staining protocol that allows for NMJs to still be analyzed even when encountered with a situation in which the use of α‐BTX is not feasible (i.e., a tissue sample has been paraffin‐embedded and can no longer be cryosectioned). In the future, a multi‐species study directly comparing standard frozen section analysis to our novel paraffin‐embedded methods is needed to fully determine the benefits and drawbacks of each methodology as they relate specifically to NMJ visualization.

5. CONCLUSION

Herein, we describe a new methodology to visualize NMJs within paraffin‐embedded TA muscle sections. Our protocol avoids the known limitations associated with cryosectioned samples and should be widely applicable for histologic analysis in animal and human studies. In conjunction with standard cryosectioned histology methods, the described paraffin‐embedded protocol serves to enhance morphologic analysis for those studying NMJs.

Supporting information

FIGURE S1. Slides confirming the distinct staining of NEFL and SV2. Additional slides were stained utilizing a rabbit‐derived neurofilament stain (ab9568) (microscope = echo revolve, zoom = 40×, scale bar = 50 μm, red = SV2, green = NF and CHRNα₁, blue = nuclei).

Kaefer SL, Shay EO, Morrison RA, Zhang L, Voytik‐Harbin S, Halum S. Neuromuscular junction visualization in paraffin‐embedded thyroarytenoid muscle sections: Expanding options beyond frozen section analysis. Laryngoscope Investigative Otolaryngology. 2025;10(1):e70020. doi: 10.1002/lio2.70020