Name: Technique of Conjunctival Biopsy and Direct Immunofluorescence for Diagnosing Mucous Membrane Pemphigoid (Video) | JoVE Journal
Uploaded: 2025-06-17T15:00:00.000+00:00
Duration: 5 min 5 s
Description: Learn conjunctival biopsy techniques for diagnosing mucous membrane pemphigoid using direct immunofluorescence staining. This standardized protocol enables accurate diagnosis to prevent blindness in patients.

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5 minutes

We thank the Hyderabad Eye Research Foundation, India for supporting this study.

The protocol follows the guidelines of the human research ethics committee of LV Prasad Eye Institute human research ethics committee.
1. Conjunctival biopsy technique
<ol>
	<li>Plan biopsy from both eyes of the patient suspected of pemphigoid under topical or local anesthesia and operating microscope.</li>
	<li>Take informed consent from the patient and list possible side effects of bleeding, scarring, and the need to start topical steroids following surgery.</li>
	<li>Place 1-2 drops of 0.5% proparacaine in both eyes and wait 5 min before starting the procedure.</li>
	<li>Ensure the patient does not feel pain when touching the conjunctiva with fine forceps. If the patient still feels pain after applying topical proparacaine, inject 0.2 mL of subconjunctival 2% lidocaine with a 1 mL tuberculin syringe.</li>
	<li>Place a speculum in the right or left eye, whichever is chosen first.</li>
	<li>Examine the ocular surface for areas of scarring and keratinization. Select the biopsy area that is uninvolved and not scarred or keratinized. 
	NOTE: In this study, the superior bulbar conjunctiva away from the limbus is preferred as the biopsy site, followed by the inferior bulbar conjunctiva.</li>
	<li>Use Lim&#39;s forceps to lift the sclera off the conjunctiva and linearly excise with Westcott&#39;s scissors, measuring 4-5 mm long and 2-3 mm wide.</li>
	<li>Transfer the whole biopsy immediately to an optimal cutting medium (OCT). 
	NOTE: One can transfer biopsies in normal saline or a Michel transport medium.</li>
	<li>Take a cartridge and a plastic tissue mould with a well in the centre, clean it with saline, and air-dry it.</li>
	<li>Fill the cartridge with OCT medium separately for the right and left eye. Submerge the excised conjunctival tissue in the OCT medium and send it to pathology.</li>
	<li>Repeat the similar steps for the contralateral side as well.</li>
</ol>
2. Transferring the tissue
<ol>
	<li>Embed the biopsy specimens of the conjunctiva in an OCT medium to preserve tissue architecture and antigenicity if received in Michel&#39;s transport medium or normal saline.</li>
	<li>Snap-freeze the embedded samples using liquid nitrogen or dry ice and store at -80 &#176;C overnight.</li>
</ol>
3. Preparation and tissue sectioning
<ol>
	<li>Section the frozen tissue into 4-6 &#181;m slices (7 in number) using a cryostat at -20 &#176;C to -25 &#176;C.</li>
	<li>Mount the sections on positively charged polylysine-coated microscope slides to ensure adhesion and store at -80 &#176;C until staining in aluminum foil.</li>
</ol>
4. Immunofluorescent staining
<ol>
	<li>Thaw slides at room temperature (RT) for 10 min and fix in cold acetone (100%) for 10 min to preserve antigens and prevent tissue damage.</li>
	<li>Rinse with 1x PBS thrice, each time for 5 min to remove excess OCT medium and acetone.</li>
	<li>Apply conjugated antibodies anti-IgG, anti-IgA, anti-IgM, and anti-C3 diluted (Fibrinogen-1:10 [positive control], IgG-1:10, IgA-1:10, IgM-1:10, C3-1:5, C4-1:10) over slides. 
	NOTE: Antibodies are diluted in 1x PBS.</li>
	<li>For negative controls, skip the application of conjugated antibodies and follow the remaining steps mentioned below.</li>
	<li>Follow the manufacturer&#39;s guidelines for diluting the antibodies and standardize with known positive and negative controls.</li>
	<li>Incubate the slides in a humidified chamber at 37 &#176;C for 45-60 min.</li>
	<li>Wash slides with PBS (3&#215; for 5 min each) to remove unbound antibodies.</li>
	<li>Counterstain with DAPI (4&#39;,6 -diamidino-2-phenylidole)-containing mounting medium for nuclear visualization.</li>
	<li>Place a coverslip over the slides and allow them to cure for 20 min.</li>
	<li>Examine slides using a fluorescence microscope equipped with appropriate filters for the fluorophores used (excitation green filter for 488 nm).</li>
	<li>Document the findings with digital micrographs.</li>
</ol>
5. Rapid periodic acid - Schiff staining
<ol>
	<li>Preparation of periodic acid Schiff (PAS)
	<ol>
		<li>Dissolve 1 g of basic fuschin in 100 mL of hot distilled water.</li>
		<li>Add 10 mL of hydrochloric acid and 2 g of potassium disulfite to decolorize the dye.</li>
		<li>After cooling, add 0.5 g of activated charcoal, stir, and filter the solution to obtain clear periodic acid Schiff&#39;s reagent. Store in a dark bottle. 
		NOTE: To test for Schiff&#39;s reagent, pour 10 mL of 37% formalin into a watch glass and add a few drops of Schiff&#39;s to be tested. A good Schiff&#39;s reagent will rapidly turn a red-purple color. A deteriorating Schiff&#39;s reagent will cause a delayed reaction, and the product will be a deep blue-purple.</li>
	</ol>
	</li>
	<li>PAS staining technique
	<ol>
		<li>Hydrate the slides in water.</li>
		<li>Oxidize in 0.5% periodic acid solution for 5 min.</li>
		<li>Rinse in distilled water.</li>
		<li>Place in Schiff&#39;s reagent for 15 min (sections look light pink during this step)</li>
		<li>Wash in lukewarm tap water for 5 min (sections turn dark pink).</li>
		<li>Counterstain in Mayer&#39;s hematoxylin for 1 min.</li>
		<li>Wash in tap water for 5 min.</li>
		<li>Dehydrate the slides in the following solutions: One time in 80% ethyl alcohol for 1 min, two times in 90% ethyl alcohol for 1 min, two times in 100% ethyl alcohol for 1 min.</li>
		<li>Dehydrate the slides three times in xylene for 1 min each.</li>
		<li>Place a coverslip over the slides using a synthetic mounting medium (e.g., DPX mountant)</li>
		<li>Observe the PAS results under the bright field microscope.</li>
	</ol>
	</li>
</ol>

<ol><li>Jain, N., Jakati, S., Shanbhag, S. S., Basu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+immunofluorescence+findings+and+factors+affecting+conjunctival+biopsy+positivity+in+ocular+mucous+membrane+pemphigoid%5BTitle%5D)+AND+%22Cornea%22%5BJournal%5D)">Direct immunofluorescence findings and factors affecting conjunctival biopsy positivity in ocular mucous membrane pemphigoid.</a> Cornea. 43 (2), 159-165 (2024).</li>
<li>Vazirani, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+cicatrizing+conjunctivitis%3A+a+review+of+the+differential+diagnosis+and+an+algorithmic+approach+to+management%5BTitle%5D)+AND+%22Indian+J+Ophthalmol%22%5BJournal%5D)">Chronic cicatrizing conjunctivitis: a review of the differential diagnosis and an algorithmic approach to management.</a> Indian J Ophthalmol. 68 (11), 2349-2355 (2020).</li>
<li>Singh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug-induced+cicatrizing+conjunctivitis%3A+a+case+series+with+review+of+etiopathogenesis%2C+diagnosis+and+management%5BTitle%5D)+AND+%22Ocul+Surf%22%5BJournal%5D)">Drug-induced cicatrizing conjunctivitis: a case series with review of etiopathogenesis, diagnosis and management.</a> Ocul Surf. 24, 83-92 (2022).</li>
<li>Bocanegra-Oyola, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+characteristics+of+ocular+mucous+membrane+pemphigoid%3A+a+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22Ocul+Immunol+Inflamm%22%5BJournal%5D)">Clinical characteristics of ocular mucous membrane pemphigoid: a systematic review and meta-analysis.</a> Ocul Immunol Inflamm. 32 (10), 2388-2404 (2024).</li>
<li>Coco, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conjunctival+biopsy+site+in+mucous+membrane+pemphigoid%5BTitle%5D)+AND+%22Am+J+Ophthalmol%22%5BJournal%5D)">Conjunctival biopsy site in mucous membrane pemphigoid.</a> Am J Ophthalmol. 216, 1-6 (2020).</li>
<li>Ong, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mucous+membrane+pemphigoid+with+ocular+involvement%3A+the+clinical+phenotype+and+its+relationship+to+direct+immunofluorescence+findings%5BTitle%5D)+AND+%22Ophthalmology%22%5BJournal%5D)">Mucous membrane pemphigoid with ocular involvement: the clinical phenotype and its relationship to direct immunofluorescence findings.</a> Ophthalmology. 125 (4), 496-504 (2018).</li>
<li>Anesi, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reliability+of+conjunctival+biopsy+for+diagnosis+of+ocular+mucous+membrane+pemphigoid%3A+redetermination+of+the+standard+for+diagnosis+and+outcomes+of+previously+biopsy-negative+patients%5BTitle%5D)+AND+%22Ocul+Immunol+Inflamm%22%5BJournal%5D)">Reliability of conjunctival biopsy for diagnosis of ocular mucous membrane pemphigoid: redetermination of the standard for diagnosis and outcomes of previously biopsy-negative patients.</a> Ocul Immunol Inflamm. 29 (6), 1106-1113 (2021).</li>
<li>Dart, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoantibody+detection+for+diagnosis+in+direct+immunofluorescence-negative+mucous+membrane+pemphigoid%3A+ocular+and+other+sites+compared%5BTitle%5D)+AND+%22Ophthalmology%22%5BJournal%5D)">Autoantibody detection for diagnosis in direct immunofluorescence-negative mucous membrane pemphigoid: ocular and other sites compared.</a> Ophthalmology. 128 (3), 372-382 (2021).</li>
<li>Leonard, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunofluorescent+studies+in+ocular+cicatricial+pemphigoid%5BTitle%5D)+AND+%22Br+J+Dermatol%22%5BJournal%5D)">Immunofluorescent studies in ocular cicatricial pemphigoid.</a> Br J Dermatol. 118 (2), 209-217 (1988).</li>
<li>Mehra, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnostic+relevance+of+direct+immunofluorescence+in+ocular+mucous+membrane+pemphigoid%5BTitle%5D)+AND+%22JDDG%22%5BJournal%5D)">Diagnostic relevance of direct immunofluorescence in ocular mucous membrane pemphigoid.</a> JDDG. 13 (12), 1268-1274 (2015).</li>
<li>Shimanovich, I., Nitz, J. M., Zillikens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+and+repeated+sampling+increases+the+sensitivity+of+direct+immunofluorescence+testing+for+the+diagnosis+of+mucous+membrane+pemphigoid%5BTitle%5D)+AND+%22J+Am+Acad+Dermatol%22%5BJournal%5D)">Multiple and repeated sampling increases the sensitivity of direct immunofluorescence testing for the diagnosis of mucous membrane pemphigoid.</a> J Am Acad Dermatol. 77 (4), 700-705 (2017).</li>
<li>Goldich, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characteristics+of+patients+with+ocular+cicatricial+pemphigoid+referred+to+major+tertiary+hospital%5BTitle%5D)+AND+%22Can+J+Ophthalmol%22%5BJournal%5D)">Characteristics of patients with ocular cicatricial pemphigoid referred to major tertiary hospital.</a> Can J Ophthalmol. 50 (2), 137-142 (2015).</li>
<li>Mudhar, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biopsies+of+cicatricial+conjunctivitis+cases+reveal+highly+variable+sampling+practice+among+ophthalmologists%3A+time+for+national+and+international+standardisation%5BTitle%5D)+AND+%22Br+J+Ophthalmol%22%5BJournal%5D)">Biopsies of cicatricial conjunctivitis cases reveal highly variable sampling practice among ophthalmologists: time for national and international standardisation.</a> Br J Ophthalmol. 100 (6), 736-744 (2016).</li>
<li>Vodegel, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+diagnostic+immunofluorescence+using+biopsies+transported+in+saline%5BTitle%5D)+AND+%22BMC+Dermatol%22%5BJournal%5D)">Enhanced diagnostic immunofluorescence using biopsies transported in saline.</a> BMC Dermatol. 4, 1-7 (2004).</li>
<li>Jonkman, M. F., De Groot, A. C., Slegers, T. P., De Jong, M. C., Pas, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+diagnosis+of+pure+ocular+mucous+membrane+pemphigoid%3A+indirect+immunofluorescence+versus+immunoblot%5BTitle%5D)+AND+%22Eur+J+Dermatol%22%5BJournal%5D)">Immune diagnosis of pure ocular mucous membrane pemphigoid: indirect immunofluorescence versus immunoblot.</a> Eur J Dermatol. 19 (5), 456-460 (2009).</li>
<li>Shimanovich, I., Nitz, J. M., Witte, M., Zillikens, D., Rose, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunohistochemical+diagnosis+of+mucous+membrane+pemphigoid%5BTitle%5D)+AND+%22J+Oral+Pathol+Med%22%5BJournal%5D)">Immunohistochemical diagnosis of mucous membrane pemphigoid.</a> J Oral Pathol Med. 47 (6), 613-619 (2018).</li>
<li>Philip, A. M., Stephenson, A., Al-Dabbagh, A., Ramezani, K., Fernandez-Santos, C. C., Foster, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ocular+cicatricial+pemphigoid+with+IgM-positive+biopsy%5BTitle%5D)+AND+%22Cornea%22%5BJournal%5D)">Ocular cicatricial pemphigoid with IgM-positive biopsy.</a> Cornea. 42 (12), 1503-1505 (2023).</li>
<li>Williams, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conjunctival+neutrophils+predict+progressive+scarring+in+ocular+mucous+membrane+pemphigoid%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Conjunctival neutrophils predict progressive scarring in ocular mucous membrane pemphigoid.</a> Invest Ophthalmol Vis Sci. 57 (13), 5457-5469 (2016).</li>
<li>Levian, B., Hussaini, S. S., Woodley, D. T., Kasperkiewicz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudopemphigoid%3A+a+systematic+review%5BTitle%5D)+AND+%22JAAD+Int%22%5BJournal%5D)">Pseudopemphigoid: a systematic review.</a> JAAD Int. 17, 170-171 (2024).</li>
</ol>

The authors have nothing to disclose.

The technique of conjunctival biopsy described here uses OCT medium as a transfer medium, obviating the need for an additional transfer medium and avoiding the biopsy handling for transferring it to OCT medium for cryosectioning. A plastic cartridge mold filled with medium and biopsy is directly frozen to make tissue blocks and then sectioned. The advantages of OCT are that it freezes rapidly with minimal damage from crystal formation and encapsulates the tissue specimens. The most critical step in direct immunofluoresce...

Mucous membrane pemphigoid (MMP) is a chronic cicatrizing conjunctival disease of autoimmune etiology and affects mucosal membranes in the body1,2. Chronic cicatrizing conjunctivitis has different etiologies, and differentiation of MMP from SJS, sarcoidosis, ocular squamous neoplasia, and pseudopemphigoid is necessary as the former requires systemic immunosuppression3,4. Moreover, MMP is a potentially blinding disease where patients often present late and are misdiagnosed initially1,2,5. Only ocular involvement without systemic involvement can be seen in 27.4% of ocular MMP patients6. Diagnosis of MMP requires a conjunctival biopsy to show the presence of autoantibodies in the basement membrane2,5. Direct immunofluorescence (DIF) is a pivotal diagnostic tool for identifying autoimmune diseases affecting the conjunctiva, such as MMP. DIF helps detect the in-situ deposition of immunoglobulins and complement components at the basement membrane zone (BMZ), a characteristic of MMP. The DIF positivity rates vary from 30% to 80%1,5. However, some patients can have a negative biopsy, especially those with ocular-only MMP, but clinical documentation of disease progression is treated similarly to biopsy-positive ocular MMP cases7,8. Hence, Dart et al. stressed the need for separate diagnostic criteria for ocular-only MMP8.
The proposed reasons for the low positivity rate across studies are advanced disease with extensive ocular surface scarring, inadequate tissue processing or staining techniques, and a biopsy-negative disease variant8,9,10,11. A retrospective analysis of biopsies from 27 patients suspected of ocular MMP revealed only a 30% positivity rate, and 63.7% had only ocular involvement12. DIF requires a specific protocol; not every diagnostic laboratory is attuned to that. There are differences regarding the biopsy site, whether to perform a biopsy from one or both eyes, and sample transfer solutions13,14,15. In a retrospective analysis of the conjunctival biopsies at our center, the DIF positivity rate was 54.1%, with more positives when biopsied bilaterally (59% vs. 45%)8. Although a biopsy is critical, clinical signs and progressive ocular surface cicatrization help clinch the diagnosis of MMP. It is easier to diagnose when an extraocular site is involved, such as oral or skin, reported in 40% and 18% of patients, respectively5.
There are variations in the existing literature, and the protocol presented here can serve as a reference guide with modifications introduced to reduce the need for a unique transfer medium14. The biopsies are transferred in an optimal cutting temperature (OCT) medium and snap-frozen in the same transfer cartridge in the pathology lab, obviating the need for an additional transfer medium. The current paper outlines the methodology for DIF staining of conjunctival tissue, tissue harvesting, and sample transfer protocol.

<table><tbody><tr><td>1 N hydrochloric acid&amp;nbsp;</td><td>Finar&amp;nbsp; chemical Limited&amp;nbsp;</td><td>7647-01-0</td><td></td></tr><tr><td>Acetone&amp;nbsp;</td><td>Qualigens</td><td>Q33517</td><td></td></tr><tr><td>Albumin</td><td>DAKO</td><td>F0117</td><td></td></tr><tr><td>Basic fuchsin</td><td>Qualigens</td><td>Q39402</td><td></td></tr><tr><td>C3c</td><td>Diagnostic Biosystems&amp;nbsp;</td><td>F003</td><td></td></tr><tr><td>C4c</td><td>Diagnostic Biosystems&amp;nbsp;</td><td>F005</td><td></td></tr><tr><td>Cartridge</td><td>Polysciences</td><td>25376-500</td><td></td></tr><tr><td>Charcoal Activated&amp;nbsp;</td><td>Sigma</td><td>C-5260</td><td></td></tr><tr><td>Cryostat&amp;nbsp;</td><td>Leica&amp;nbsp;</td><td>CM1860UV&amp;nbsp;</td><td></td></tr><tr><td>Cryotome blades&amp;nbsp;</td><td>Leica&amp;nbsp;</td><td>818</td><td></td></tr><tr><td>DAPI</td><td>Abcam&amp;nbsp;</td><td>Ab104139</td><td></td></tr><tr><td>Fibrinogen&amp;nbsp;</td><td>Diagnostic Biosystems&amp;nbsp;</td><td>F006</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Olympus</td><td>CX43</td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Joja surgical</td><td>JS1528A</td><td></td></tr><tr><td>Humidity Chamber&amp;nbsp;</td><td>Diagnostic Biosystems&amp;nbsp;</td><td>SM365</td><td></td></tr><tr><td>IgA</td><td>Diagnostic Biosystems&amp;nbsp;</td><td>F007</td><td></td></tr><tr><td>IgG</td><td>Diagnostic Biosystems&amp;nbsp;</td><td>F008</td><td></td></tr><tr><td>IgM</td><td>Diagnostic Biosystems&amp;nbsp;</td><td>F009</td><td></td></tr><tr><td>IHC Adhesive glass slides&amp;nbsp;</td><td>Tomo&amp;nbsp;</td><td>33140455</td><td></td></tr><tr><td>Lim&#039;s forceps</td><td>Speedway&amp;nbsp;</td><td>S-7223</td><td></td></tr><tr><td>Michel&#039;s tansport medium</td><td>Fisher Scinitics&amp;nbsp;</td><td>NC0253304</td><td></td></tr><tr><td>Microscopic cover glass</td><td>Blue star</td><td>https://www.bluestarslides.com</td><td></td></tr><tr><td>OCT Medium&amp;nbsp;</td><td>Cryomatrix&amp;nbsp;</td><td>6769006</td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>DAKO</td><td>dm831</td><td></td></tr><tr><td>Periodic acid&amp;nbsp;</td><td>Sigma</td><td>375810-25G</td><td></td></tr><tr><td>Potassium disulfite</td><td>Sigma</td><td>P2522-500G</td><td></td></tr><tr><td>Proparacaine 0.5%</td><td>Sunway Private&amp;nbsp;</td><td>G/1118A</td><td></td></tr><tr><td>Westcott&#039;s scissors</td><td>Pillig Gurgical Intruments&amp;nbsp;</td><td>423480</td><td></td></tr></tbody></table>

technique of conjunctival biopsy and direct immunofluorescence for diagnosing mucous membrane pemphigoid

Technique of Conjunctival Biopsy and Direct Immunofluorescence for Diagnosing Mucous Membrane Pemphigoid

Ocular mucous membrane pemphigoid (MMP) presents as chronic cicatrizing conjunctivitis that, if left untreated, can cause blindness. Timely diagnosis and immunosuppressive therapy prevent disease progression. Direct immunofluorescence staining (DIF) of the conjunctival biopsy for immune complex deposition in the basement membrane is diagnostic of MMP. The biopsy positivity rate depends on biopsy characteristics, laterality, old age, and disease activity. The current methods paper describes the technique of harvesting the conjunctival biopsy and using optimal cutting temperature medium-filled cartridges for sample transfer and block preparation. A biopsy is performed from a non-scarred, uninvolved, or relatively less-involved region of the conjunctiva in both eyes. The usual biopsy size is 4-5 mm long, 2-3 mm wide, and should have tiny underlying subconjunctival tissue so that the basement membrane is not missed. We recommend performing periodic acid Schiff staining to confirm the presence of the basement membrane. Using a specified protocol, the cryosections from biopsies are stained with fluorescent-conjugated antibodies IgG, IgM, IgA, and complement factors C3c and C4c. A positive biopsy shows the green positive fluorescence in the basement membrane, and even if one of the antibodies is positive, it is MMP positive. A negative biopsy does not rule out pemphigoid; however, a positive one confirms the MMP and differentiates it from drug-induced cicatrizing conjunctivitis or pseudopemphigoid.

The interpretation of DIF requires confirmation of the presence of the basement membrane in the tissue sections (Figure 1). In the PAS-stained slides, the basement membrane will appear as a bright pink membrane situated at the basal region of the conjunctival epithelium (Figure 1C). The basement membrane's continuity and architecture should also be commented upon. DIF positivity will be seen as bright green BM (Figure 1B), indicatin...

Here, we present a protocol for harvesting and processing conjunctival biopsy to identify mucous membrane pemphigoid.

Ocular mucous membrane pemphigoid (MMP) presents as chronic cicatrizing conjunctivitis that, if left untreated, can cause blindness. Timely diagnosis and ...

We are trying to increase the adoption of conjunctival biopsy for the diagnosis of mucous membrane pemphigoid, which is a chronic cicatrizing conjunctivitis, and if left untreated, it can cause blindness. Direct immunofluorescent staining of the conjunctival biopsy for immune complex deposition in the patient membrane is diagnostic of MMP, but is not performed at many centers across the world. The current methods paper describe the techniques of the harvesting conjunctival biopsy and using optimal cutting temperature medium filled with cartridges for sample transfer and blood preparation, which reduce the sample handling.To begin, plan a biopsy from both eyes of the patient suspected of pemphigoid under topical or local anesthesia using an operating microscope. Take informed consent from the patient and clearly explain the possible side effects, including bleeding, scarring, and the potential need to start topical steroids after surgery. Before taking a biopsy, prepare the cartridges by cleaning them using normal saline and filling with optimal cutting temperature, or OCT medium separately for each eye.Now place a speculum into the selected eye, either right or left. After applying topical proparacaine, if the patient still experiences pain, inject 0.2 milliliters of 2% lidocaine subconjunctivally using a 1 milliliter to tuberculin syringe. After lifting the conjunctiva off the sclera using limbs forceps, perform a linear excision measuring approximately 4-5 millimeters long and 2-3 millimeters wide with a pair of Westcott scissors.Immediately transfer the entire biopsy into an optimal cutting temperature medium. Snap freeze the embedded samples using liquid nitrogen or dry ice. Then store them at -80 degrees Celsius overnight.Section the frozen tissue into 4-6 micrometer thick slices using a cryostat maintained at -20 to -25 degrees Celsius. Mount the sections onto positively charged polylysine-coated microscope slides to ensure adhesion. Then wrap the slides in aluminum foil before storing at -80 degrees Celsius until staining.Thaw the slides at room temperature for 10 minutes. Then fix the tissue in 100%cold acetone for another 10 minutes to preserve antigens and minimize tissue damage. Now, rinse the slides with OBS three times, each for five minutes to eliminate residual OCT medium and acetone.Apply diluted conjugated antibodies, anti-IgG, anti-IgA, anti-IgM, and anti-IgC3 over the slides. Do not apply the antibodies for the negative control slide. Now incubate the slides in a humidified chamber at 37 degrees Celsius for 45-60 minutes.Then wash the slides with PBS three times for five minutes each to remove unbound antibodies. Counterstain with a mounting medium containing DAPI for nuclear visualization. Lastly, place a cover slip over each slide and allow them to cure for 20 minutes.Examine the slides using a fluorescence microscope equipped with the appropriate filters, including a green excitation filter at 488 nanometers for fluorophore detection. The basement membrane was confirmed in conjunctival biopsies using PAS staining, appearing as a continuous bright pink band at the base of the epithelium. Direct immunofluorescence positivity was visualized as bright green fluorescence along the basement membrane.Direct immunofluorescence positive biopsies showed fluorescence for immunoglobulins IgM, IgA and IgG, while negative biopsies lacked fluorescence. The positive control showed strong green fluorescence at the basement membrane.

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The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Studying tumor microenvironment may identify prognostic or predictive biomarkers of clinical response to immunotherapy. Presented here, is an innovative method based on in situ fluorescence multispectral imaging to analyze and count automatically various subpopulations of CD8+ T cells. This reproducible and reliable technique is suitable for large cohort analyses.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Skin lesions are common and important features in many rheumatic diseases, and biopsy with pathological analysis plays a critical role in diagnosis. This article provides a detailed overview of the standardized skin biopsy procedure and, using discoid lupus erythematosus as an example, highlights the essential role of dermatopathology in diagnosing rheumatic diseases.

Skin Biopsy for Diagnosing Discoid Lupus Erythematosus

This article demonstrates the operation and effect of in vivo confocal microscopy in the diagnosis and treatment of dry eye and investigates the application and outcomes of in vivo confocal microscopy to provide a basis for future research.

In Vivo Confocal Microscopy in the Diagnosis and Management of Dry Eye: A Focus on Imaging Protocols and Interpretation

Learn conjunctival biopsy techniques for diagnosing mucous membrane pemphigoid using direct immunofluorescence staining. This standardized protocol enables accurate diagnosis to prevent blindness in patients.

Technique of Conjunctival Biopsy and Direct Immunofluorescence for Diagnosing Mucous Membrane Pemphigoid

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