Name: Dos and Donts in the Preparation of Muscle Cryosections for Histological Analysis (Video) | JoVE Journal
Uploaded: 2015-05-15T13:00:00.000+00:00
Duration: 10 min 27 s
Description: Learn to prepare muscle cryosections for histological analysis using rapid freezing in methyl butane and OCT embedding. This method preserves tissue integrity and prevents freezing artifacts for accurate evaluation.

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5 minutes

We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1. Tissue Harvest and Freezing of Muscle Tissues
<ol>
	<li>Euthanize the mouse with an overdose of isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane). Perform this step in a fume hood and use a secondary containment method such as a desiccator or a bell jar containing a cotton swab soaked in isoflurane.</li>
	<li>Confirm death by firmly squeezing footpad. Use a secondary method of euthanasia such as cervical dislocation.</li>
	<li>Wet the fur with 70% alcohol to reduce the sticking of loose hair strands on the muscles. Peel the skin off the limb using a fine forceps such as #5.</li>
	<li>Carefully separate the muscle of interest (tibialis anterior (TA) in this case) from the bones underneath, starting from the tendon and remove.</li>
	<li>Excise the muscle, cover it in OCT (Optimum Cutting Temperature compound), and lay it flat on a labeled disposable freezing mold. Make sure that the muscle is at its normal physiological orientation (head to tail) without stretching. Alternatively, freeze the muscle on small styrofoam squares and pin it using insect pins to ensure the correct length of the muscles.</li>
	<li>Chill 2-methylbutane (isopentane) in a steel beaker using liquid nitrogen. This takes on average 3 - 5 min. Keep stirring intermittently until white precipitates start to form on the bottom and walls of the beaker. 
	NOTE: When this occurs, isopentane has reached optimal freezing temperature (-150 &#176;C)</li>
	<li>Dip the molds containing muscles in the chilled 2-methylbutane. Freeze small muscles such as the diaphragm and extensor digitorum longus (EDL) for 6 - 12 sec and larger muscles like the gastrocnemius soleus (GS) and triceps for 15 - 20 sec. Do not freeze the muscles for too long as this may lead to cracking.</li>
	<li>Transfer the frozen tissues on to dry ice and let the isopentane evaporate (note that this process takes on average around 15 - 20 min). Wrap the tissues in aluminum foil and store them at ultra low temperature (-70 &#176;C to -80 &#176;C) until sectioning.</li>
</ol>
2. Embedding the Tissues in OCT to Prepare Tissue Block
<ol>
	<li>Transport tissues on dry ice to cyrostat to prevent thawing. Transfer tissue to cryostat chamber (set at -20 to -24 &#176;C to avoid full thawing of tissues) and let them equilibrate for 30 min.</li>
	<li>Turn the Peltier bay cooling on. If the cryostat does not have this cooling, use aerosol cooling spray to quickly freeze the OCT. Do not let the muscle thaw at any point during the embedding as this can result in freeze damage.</li>
	<li>Add a uniform thin layer of OCT on the specimen disc and let it cool. When most of the OCT is frozen on the bottom while still liquid on the top, insert the tissue at the correct orientation (perpendicular to OCT for transverse sections and parallel to OCT for longitudinal sections). Use aerosol cooling spray to quickly freeze OCT. Touch the unembeded part of the tissue with heat extractor and wait for 45 sec.</li>
	<li>Add another thin layer of OCT around the muscle, spray with aerosol cooling spray and extract the heat as in the previous step.</li>
	<li>Keep adding OCT in small increments and quickly freeze the OCT using a combination of aerosol cooling spray and heat extractor until the whole muscle is covered.</li>
	<li>Put the heat extractor on the top of the tissue block and wait for 5 min.</li>
</ol>
3. Preparing Sections and Identifying the Sections from Mid-belly Region of the Muscles
<ol>
	<li>Mount the specimen onto the specimen head. Tighten the knob to secure the disc. Make sure that the specimen disc is in good contact with the specimen head.</li>
	<li>Adjust the chamber temperature to between -21 and -24 &#176;C and wait until the set temperature is achieved.</li>
	<li>Adjust the thickness settings (7 &#181;m); trim the block by advancing or retracting the block using coarse and fine settings.</li>
	<li>Collect the sections on pre-warmed positively charged microscope slides by pressing the top of the slide onto the sections, using the attraction between opposite charges to facilitate the adherence of the cut sections to the slide.</li>
	<li>Identify the mid-belly sections by measuring cross-sectional area (CSA) of the sections. Do this using imaging software on the computer that is connected to the microscope. 
	NOTE: Phase contrast images are taken and circumference of the entire muscle is measured via a tracing function on the software program. This will yield both CSA as well as minimum ferret diameter (measure of fiber cross-sectional size that is expressed as the smallest distance between two parallel tangents on opposite sides of a particle) measurements. Note: When CSA and minimum ferret diameter measurements begin to plateau, mid belly has been reached.</li>
	<li>Inventory and archive the slides at -80 &#176;C for future staining. 
	NOTE: OCT does not need to be removed prior to staining procedures. Once slides are coverslipped, they are ready to be stained immediately.</li>
</ol>
4. Staining
NOTE: While detailed step-by-step procedures can be found on the product data sheet and should be followed for staining procedures, personal optimization may be required to obtain optimal staining.
<ol>
	<li>Because mounting media (see table) is not miscible with water, dehydrate slides through increasing grades of alcohol (2 min each in 70%, 95%, and 100% ethanol) and clear&#160;them with xylene (100% for 5 min) to ensure slides do not become opaque over time.</li>
	<li>Coverslip sections and let them dry. Image with microscope equipped with imaging software.</li>
</ol>

<ol><li>Bonnemann, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnostic+approach+to+the+congenital+muscular+dystrophies%5BTitle%5D)+AND+%22Neuromuscular+disorders+%3A+NMD%22%5BJournal%5D)">Diagnostic approach to the congenital muscular dystrophies.</a> Neuromuscular disorders : NMD. 24, 289-311 (2014).</li>
<li>Caughey, J. E., Pachomov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+diaphragm+in+dystrophia+myotonica%5BTitle%5D)+AND+%22J+Neurol+Neurosurg+Psychiatry%22%5BJournal%5D)">The diaphragm in dystrophia myotonica.</a> J Neurol Neurosurg Psychiatry. 22, 311-313 (1959).</li>
<li>Fetterman, G. H., Wratney, M. J., Donaldson, J. S., Danowski, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscular+dystrophy.+I.+History%2C+clinical+status%2C+muscle+strength%2C+and+biopsy+findings%5BTitle%5D)+AND+%22AMA+J+Dis+Child%22%5BJournal%5D)">Muscular dystrophy. I. History, clinical status, muscle strength, and biopsy findings.</a> AMA J Dis Child. 91, 326-338 (1956).</li>
<li>Platzer, A. C., Chase, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histologic+Alterations+in+Preclinical+Mouse+Muscular+Dystrophy%5BTitle%5D)+AND+%22The+American+journal+of+pathology%22%5BJournal%5D)">Histologic Alterations in Preclinical Mouse Muscular Dystrophy.</a> The American journal of pathology. 44, 931-946 (1964).</li>
<li>Mercuri, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+magnetic+resonance+imaging+involvement+in+muscular+dystrophies+with+rigidity+of+the+spine%5BTitle%5D)+AND+%22Ann+Neurol%22%5BJournal%5D)">Muscle magnetic resonance imaging involvement in muscular dystrophies with rigidity of the spine.</a> Ann Neurol. 67, 201-208 (2010).</li>
<li>Tasca, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Different+molecular+signatures+in+magnetic+resonance+imaging-staged+facioscapulohumeral+muscular+dystrophy+muscles%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Different molecular signatures in magnetic resonance imaging-staged facioscapulohumeral muscular dystrophy muscles.</a> PloS one. 7, e38779(2012).</li>
<li>Girgenrath, M., Dominov, J. A., Kostek, C. A., Miller, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+apoptosis+improves+outcome+in+a+model+of+congenital+muscular+dystrophy%5BTitle%5D)+AND+%22The+Journal+of+clinical+investigation%22%5BJournal%5D)">Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy.</a> The Journal of clinical investigation. 114, 1635-1639 (2004).</li>
<li>Kumar, A., Yamauchi, J., Girgenrath, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle-specific+expression+of+insulin-like+growth+factor+1+improves+outcome+in+Lama2Dy-w+mice%2C+a+model+for+congenital+muscular+dystrophy+type+1A%5BTitle%5D)+AND+%22Human+molecular+genetics%22%5BJournal%5D)">Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A.</a> Human molecular genetics. 20, 2333-2343 (2011).</li>
<li>Mehuron, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dysregulation+of+matricellular+proteins+is+an+early+signature+of+pathology+in+laminin-deficient+muscular+dystrophy%5BTitle%5D)+AND+%22Skeletal+muscle%22%5BJournal%5D)">Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy.</a> Skeletal muscle. 4, 14(2014).</li>
<li>Yamauchi, J., Kumar, A., Duarte, L., Mehuron, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Triggering+regeneration+and+tackling+apoptosis%3A+a+combinatorial+approach+to+treating+congenital+muscular+dystrophy+type+1+A%5BTitle%5D)+AND+%22Human+molecular+genetics%22%5BJournal%5D)">Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A.</a> Human molecular genetics. 22, 4306-4317 (2013).</li>
<li>Sheriffs, I. N., Rampling, D., Smith, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paraffin+wax+embedded+muscle+is+suitable+for+the+diagnosis+of+muscular+dystrophy%5BTitle%5D)+AND+%22Journal+of+clinical+pathology%22%5BJournal%5D)">Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy.</a> Journal of clinical pathology. 54, 517-520 (2001).</li>
<li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formaldehyde+fixation%5BTitle%5D)+AND+%22Journal+of+Histochemistry+%26+Cytochemistry%22%5BJournal%5D)">Formaldehyde fixation.</a> Journal of Histochemistry & Cytochemistry. 33, 845-853 (1985).</li>
<li>Maccarty, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Diagnostic+Reliability+of+Frozen+Sections%5BTitle%5D)+AND+%22The+American+journal+of+pathology%22%5BJournal%5D)">The Diagnostic Reliability of Frozen Sections.</a> The American journal of pathology. 5, 377-380 (1929).</li>
<li>Shi, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+value+of+frozen+tissue+section+used+as+%27gold+standard%27+for+immunohistochemistry%5BTitle%5D)+AND+%22Am+J+Clin+Pathol%22%5BJournal%5D)">Evaluation of the value of frozen tissue section used as 'gold standard' for immunohistochemistry.</a> Am J Clin Pathol. 129, 358-366 (2008).</li>
<li>Turan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accuracy+of+frozen+sections+for+intraoperative+diagnosis+of+complex+atypical+endometrial+hyperplasia%5BTitle%5D)+AND+%22Asian+Pacific+journal+of+cancer+prevention+%3A+APJCP%22%5BJournal%5D)">Accuracy of frozen sections for intraoperative diagnosis of complex atypical endometrial hyperplasia.</a> Asian Pacific journal of cancer prevention : APJCP. 13, 1953-1956 (2012).</li>
</ol>

The authors have nothing to disclose.

Histology and immunohistochemistry have been used as key tools in understanding the manifestation and progression of pathology in various neuromuscular disorders. Classically, muscle biopsies were first fixed in formaldehyde and then embedded with paraffin wax. While formaldehyde fixation preserves tissue architecture and allows tissue storage and transport at RT, it also inactivates enzymes and crosslinks proteins necessitating further steps to achieve accurate immunostaining. This problem is eliminated with the use of ...

Examination of muscle histology plays a critical role in the understanding of different types of neuromuscular disorders1-4. When combined with other techniques, it allows researchers to get a better idea of the manifestation and progression of pathology and can also be essential to evaluate the efficacy of a therapeutic intervention5-10. However, to be accurate, the tissues need to be preserved in the proper manner so as to preserve the physiological state immediately before excision. This requires great care during removal, preservation, sectioning, and staining.
Tissues can be prepared in two different ways for light microscopic study. The first method, formalin fixed paraffin embedded (FFPE) sections, requires tissues to be fixed in 10% natural buffered formaldehyde before being embedded with paraffin wax11,12. The fixation step helps to preserve morphology better but also cross links endogenous proteins thus necessitating intricate antigen retrieval procedures for immunostaining and eliminating the possibility of enzymatic staining on such sections12. Frozen sections, on the other hand, do not need to be pre-fixed or processed; therefore, proteins are able to retain native biological conformations13,14. Further, frozen sections also allow for quick turnaround time, which at times is necessary, for example in the intraoperative diagnosis of sarcomas and other surgical biopsies15. However, if not done properly, this method is prone to freeze damage, which introduces changes to muscle architecture making the sections unfit for histological examination. This paper will outline the most effective method to prepare muscle biopsies in order to achieve optimal staining for proper examination.

<table><tbody><tr><td>Cryostat</td><td>Leica CM 1850</td><td>Leica Microsystems Inc.</td><td>Buffalo Grove, IL 60089 U.S.A., office phone 1-800-248-0123&lt;br /&gt;Alternative: Richard Allen Scientific</td></tr><tr><td>Microscope</td><td>Nikon Eclipse 50i</td><td>Nikon Instruments Inc.</td><td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064 U.S.A., Phone: 1-631-547-8500&lt;br /&gt;Alternative: Olympus</td></tr><tr><td>Image analysis software</td><td>Nikon Imaging Software Basic Research</td><td>Nikon Instruments Inc.</td><td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A., Phone: 1-631-547-8500&lt;br /&gt;Alternatives: Image J, Metamorph</td></tr><tr><td>Isoflurane</td><td>14043-220-05</td><td>JD Medical Dist. Co., Inc.</td><td>1923 West Peoria Avenue, Phoenix, AZ 85029 U.S.A.</td></tr><tr><td>OCT</td><td>4583</td><td>Sakura Inc.</td><td>Torrence, CA 90501 U.S.A.&lt;br /&gt;Alternative source: Leica Microsystem</td></tr><tr><td>Freeze It</td><td>23-022524</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA, 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Disposable tissue mold</td><td>22-363-554</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Colorfrost plus slides</td><td>9991001</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Acetone</td><td>650501-4L</td><td>Sigma-Aldrich</td><td>3050 Spruce Street, St. Louis, MO 63103 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Ethyl alcohol</td><td>HC-1300-1GL</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>2-methyl butane</td><td>M 32631-SL</td><td>Sigma-Aldrich</td><td>3050 Spruce Street, St. Louis, MO 63103 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Xylene</td><td>HC-7001GA</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Cytoseal 280</td><td>8311-4</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Hematoxylin Gill #3</td><td>CS402-1D</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Eosin</td><td>6766007</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr></tbody></table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

The set-up for harvesting and freezing of tissues can be seen in Figure 1. Excised tissues should be stored on a gauze soaked in PBS to avoid drying (Figure 1A). When tissues are selected to be used for histological analysis they should be dipped in OCT and laid out on cryo mold in the proper orientation and as close to physiological length as possible to ensure accurate morphology (Figure 1B). Slurry of isopentane suitable for tissue freezing can be achieved by cooling ...

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

The overall goal of this procedure is to avoid pitfalls when preparing frozen sections from muscle biopsies, such as creating freezing artifacts. This is accomplished by first freezing the muscle rapidly in two methyl butane chilled on liquid nitrogen. After the muscle biopsies equilibrate in the cryostat, they are embedded in optimum cutting temperature, medium at room temperature in small increments with quick freezing using aerosol cooling and heat extraction.Next seven micron sections are sliced and transferred to warm, positively charged glass slides. The final step is to stain the tissue sections as needed to evaluate the tissue's histology. Ultimately correctly prepared muscle biopsies result in well-preserved tissue histology without freezing artifacts.So the main advantage of this technique over other existing methods, such as paraffin embedded tissue sectioning, is that it's very quick and it does not alter endogenous antigen structure or function. This method can help answer key questions in the health and disease of muscle biology, such as growth, regeneration, inflammation, fibrosis, and necrosis. The implication of this technique extend towards therapy or diagnosis of muscle disorders because it allows determination of condition of healthy and diseased muscle.Though this method can provide insights into different forms of muscular dystrophy, it can also be applied to other systems such as cachexia associated with terminal stages of cancer and aids and loss of muscles in elderly populations. Begin with euthanizing a mouse with an overdose of isof fluorine under a fume hood and in a secondary level of containment, such as a bell jar. Confirm the animal's death by firmly squeezing its footpad.Now, wet the fur with 70%alcohol. This prevents loose hair strands from sticking to the muscles. Proceed by peeling the skin off the limb using a number five fine forceps.Carefully separate the muscle of interest from the bone starting from the tendon. In this case, the tibias anterior is isolated. After excising the muscle, cover it in OCT and lay it flat on a labeled disposable freezing mold.More OCT will be added later before slicing. Ensure that the muscle is in its normal physiological orientation along the head to tail axis and that the muscle isn't stretched. Now chill.Some isop pentane in a steel beaker using liquid nitrogen. Stir it intermittently over three to five minutes with a plastic spatula. When a white precipitate forms the isop pentane is at its optimal temperature at this point, dip the loaded molds into the solution.Small muscles such as the diaphragm and extensor digitorum longest requires six to 12 seconds. Larger muscles require 15 to 20 seconds over freezing can lead to cracking. The next step is to put the frozen tissues on dry ice to let the residual isop pentane evaporate, which takes about 15 to 20 minutes.Once the tissue is dry, rapid in foil and store it in a minus 80 degrees Celsius freezer until it can be sectioned. Prior to sectioning. Transport the frozen tissues on dry ice to the cryostat.Transfer the tissue to a cryostat chamber set between negative 24 and negative 20 degrees Celsius, and let them equilibrate for 30 minutes. The tissues will not fully thaw at this temperature. Next, embed the tissue first.If available, turn on the Peltier Bay cooling. Second, apply a uniform thin layer of OCT on the specimen disc and let it cool. Third, once the OCT has frozen on the bottom but is still liquid on the top, insert the tissue, orient the tissue perpendicular to the OCT for transverse sections and parallel to the OCT for longitudinal sections.Now use an aerosol cooling spray to quickly freeze the OCT. Then touch the uninvented part of the tissue with a heat extractor and hold it to the tissue for 45 seconds. Do not let the muscle thaw at any step during this process.If the cryostat does not have peltier bay cooling, use aerosol cooling to quickly freeze the OCT around the tissue. Now, add another thin layer of OCT around the muscle spray with aerosol cooling spray and extract the heat as in the previous step. Keep adding OCT in small increments and quickly freeze the OCT using a combination of aerosol cooling spray and heat extractor until the whole muscle is covered.Then put the heat extractor on the top of the tissue block and wait for five minutes before proceeding with sectioning. The specimen is now ready to mount on the specimen head. First, tighten the knob to secure the disc.Make sure that the specimen disc is in good contact with the specimen head while the chamber gets to be between negative 24 and negative 20 degrees Celsius. Set the slice thickness to seven microns when the chamber is at temperature, start trimming the block. Use the course and fine adjustment to move the block.When sections are ready to collect, press their top sides onto lamp warmed positively charged slides. If frozen correctly, the tissue face should be chalky white. However, a tissue that was frozen, improperly or thawed during the mounting process will appear red to pink.The slides can be stored in the deep freezer for future analysis. The mid belly section of the tissue can be identified by measuring the cross section area from phase contrast images using the tracing function of an imaging software package. This analysis provides both area and the minimum ferret diameter measurements.When these measurements begin to plateau, the mid belly has been reached. While manufacturers provide step-by-step instructions for most staining protocols, personal optimization may be required to achieve optimal staining intensities. The OCT does not need to be removed.For staining, for optimal immuno staining, be sure to use a humidified chamber. It should be covered with perfil kept on a wet paper towel and sealed in a small box to avoid drying of sections. During the staining process for non immuno stained permanent mount slides, following the staining protocol, dehydrate the slides through increasing grades of alcohol.Because mounting media is not missable with water, then clear the slides with a five minute bath in 100%xylene. This ensures that the slides do not become opaque over time. Then proceed with mounting and imaging sections of the mid belly region of the tibials anterior were stained.Using hematin and eoin. Sections that are properly processed will have consistent morphology and even staining throughout improperly processed muscles that are freeze damaged or prematurely. Thawed appear to have multiple breaks in morphology and have the appearance of shredded wheat.Immunohistochemistry was also performed on these sections. These stains were satisfactory. Fibronectin is stained in green and dappy is seen in blue.After watching this video, you should have a thorough understanding of how to prepare a good frozen section from muscle biopsies for evaluation of muscle architecture. Once mastered, this technique can be done in approximately four to five hours if all these steps are done properly. While attempting this procedure, it is important to remember to prevent thawing of muscles Following this procedure.Other methods like immuno staining and insight to hybridization can be performed in order to answer additional questions like growth and regeneration.

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Research

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Medicine

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Learn to prepare muscle cryosections for histological analysis using rapid freezing in methyl butane and OCT embedding. This method preserves tissue integrity and prevents freezing artifacts for accurate evaluation.

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

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