Protocols Online https://www.protocolsonline.com Current and New Protocols for Life Science Research Wed, 19 Dec 2018 16:17:41 +0000 en-US hourly 1 https://www.protocolsonline.com/wp-content/uploads/2018/04/cropped-favicon-32x32.png Protocols Online https://www.protocolsonline.com 32 32 Methyl Green Counterstain Protocol https://www.protocolsonline.com/histology/dyes-and-stains/methyl-green-counterstain-protocol/ Wed, 19 Dec 2018 16:08:44 +0000 http://protocolsonline.com/?p=1430   Full Article… ]]> Introduction

Methyl green is used commonly with bright-field microscopes to stain cell nuclei by dyeing the chromatin of cells  with a green color. It is chemically related to cresyl violet.

Materials

0.1M Sodium Acetate Buffer, pH4.2

Sodium acetate, trihydrate (MW 136.1) —— 1.36 g
Distilled water ———————————– 100 ml
Mix to dissolve and adjust pH to 4.2 using concentrated glacial acetic acid

Methyl Green Solution (0.5%)

Methyl green (ethyl violet free from Sigma) —- 0.5 g
0.1M Sodium acetate buffer, pH4.2 ————- 100 ml
Mix to dissolve.

Staining Pattern

Nuclear

Suggested Use

Counterstaining for immunohistochemistry or other special stains.

Staining Procedure

  1. Sections to distilled water after IHC.
  2. Stain in methyl green solution for 5 minutes at room temperature (60 C may produce slightly stronger stain).
  3. Rinse in distilled water (sections will look blue).
  4. Dehydrate quickly through 95% alcohol (10 dips, sections turn green)
  5. 2 changes of 100% alcohol (10 dips each) (alcohol used for dehydration removes some of the stain).
  6. Clear in xylene or xylene substitute.
  7. Mount with resinous mounting medium.
]]> DAB-Peroxidase Substrate Solution (Brown) https://www.protocolsonline.com/histology/immunohistochemistry-histology/dab-peroxidase-substrate-solution-brown/ Tue, 31 Jul 2012 14:05:53 +0000 http://protocolsonline.com/?p=1426 Introduction

3,3′-Diaminobenzidine (DAB), a derivative of benzene, is an organic compound that is used in the staining of nucleic acids and proteins, most commonly for immunohistochemical procedures. Although it is water soluble in its unoxidized form, it forms a water-insoluble brown precipitate when oxidized. In immunohistochemical procedures, the location of proteins can be detected using DAB as a substrate. A protein of interest is  targeted by a antibody that is is conjugated with a peroxidase enzyme, and in the presence of hydrogen peroxide, DAB is readily catalyzed to its oxidized form, forming a brown precipate.

DAB is a mutagenic compound and should  be handled with care, using gloves and appropriate protective gear. Unused compound can be disposed of as a hazardous waste or neutralized through a simple chemical procedure.

This protocol is a simple method for preparing a basic DAB solution, producing a brown stain, for routine immunohistochemistry. Common counterstains for DAB include methyl green, hematoxylin, and Nissl stains.

Materials

  • 3,3’-diaminobenzidine. DAB-tetrahydrocholoride may be used as a substitute.
  •  30% Hydrogen Peroxide

Preparation of Stock Solutions

1% DAB (20X)

  1. Add 0.1g DAB in 10 ml of distilled water.
  2. Add 3 to 5 drops of 10N HCL to lower the pH.
  3. Shake or vortex solution until DAB completely dissolves, about 10 minutes; the solution should turn a light brown color. The solubility of DAB requires an acidic pH; lower the pH if DAB does not dissolve.
  4. Aliquot the solution and store at -20C.

0.3% Hydrogen Peroxide (20X)

  1. Add 100 ul of 30% H2O2 in 10 ml distilled water.
  2. Aliquot solution and store at -20C.

0.01M Phosphase Buffered Saline, 1 liter

  1. Add 1.20 g Na2HPO4 (anhydrous), 0.22 g NaH2PO4 · H20, and 8.5 g NaCl to 1 L of distilled water.
  2. Mix well until all reagents are dissolved.
  3. Adjust pH to 7.2 with 0.1 N HCl or 1N NaOH as needed.

Procedure

DAB-Peroxidase Substrate Solution  (0.05% DAB, 0.015% H2O2, 0.01M PBS, pH 7.2)

A critical parameter of  the DAB-Peroxidase Solution is to ensure that the pH of the solution buffered around 7.2. A pH below 7.0 will result in weak DAB intensity; a pH above 7.6 will result in a high level of background stain.

  1. Add 250 ul of 1% DAB, and 250 ul of 0.3% Hydrogen Peroxide to 5 ml of PBS.
  2. Mix well.
  3. Add enough solution to cover tissue sections, and incubate at room temperature for 1-3 minutes.
]]> Avidin-Biotin Blocking Solution https://www.protocolsonline.com/histology/immunohistochemistry-histology/avidin-biotin-blocking-solution/ Tue, 24 Jul 2012 23:31:47 +0000 http://protocolsonline.com/?p=1421 Introduction

Biotin-avidin labeling is  commonly used as a method for improving staining intensity in immunohistchemical techniqes. However, a high non-specific background is a common problem with this approach, since many tissues contain abundant quantities of biotin-binding proteins or  nonspecific binding sites. One approach to reduce non-specific background is to pre-treat tissue sections with  solutions of avidin followed by biotin. Tissues from kidney, liver, and spleen usually contain a high level of endogenous biotin, requiring a biotin-avidin block.

In cases where it is not clear if an biotin-avidin block is required, tissues can be tested using a simple procedure. Endogenous peroxidase is first neutralized through a hydrogen peroxide blocking step, and then tissue is  incubation with strepavidin-HRP followed by visualization with DAB.

Materials

Avidin in Phosphate Buffered Saline

  • Prepare 0.001% avidin (10 U/mg or greater) in  phosphate buffered saline (PBS).
  • Store at 4oC.

Biotin in Phosphate Buffered Saline

  • Prepare 0.001% biotin  in  phosphate buffered saline (PBS).
  • Store at 4oC.

Procedure

  1. Following incubation with a blocking serum, incubate with avidin for 15 minutes.
  2. Briefly immerse in wash solution.
  3. Incubate with biotin solution for 15 minutes.
  4. Briefly immerse in in wash solution to eliminate residual biotin.
  5. Proceed with addition of primary antibody for immunohistochemistry.

Rapid Avidin-Biotin Blocking Procedure

In many cases, a rapid avidin-biotin blocking procedure can be used in place of the stepwise blocking procedure. This approach combines avidin-biotin blocking with the serum and primary antibody incubation steps. It should not be used if the  primar antibody is biotinylated.

  1. Add 150 ul of avidin solution per ml of  serum block.
  2. Following serum block, briefly rinse tissue in wash solution.
  3. Add 150 ul of  biotin solution per ml of primary antibody solution, and proceed with primary antibody incubation.
]]> SDS Antigen Retrieval Solution https://www.protocolsonline.com/histology/immunohistochemistry-histology/sds-antigen-retrieval-solution/ Tue, 24 Jul 2012 23:00:02 +0000 http://protocolsonline.com/?p=1416 Introduction

A SDS antigen retrieval solution can be used on formalin or paraformaldehyde-fixed  tissues that have been sectioned on a cryostat;   typically such tissues are cryoprotected using 30% sucrose [1].  A brief exposure to SDS has been reported to greatly improve staining for some proteins.

Materials

1% Sodium Dodecyl Sulfate (SDS) in Phosphate Buffered Saline

  • Mix 1.0 g of SDS in 100 ml of  0.01 M phosphate buffered saline (PBS).
  • Store at room temperature for up to 3 months; for extended storage, store at 4oC.

Procedure

  1. Rinse sections three times for 5 minutes each in PBS.
  2. Cover sections with 1% SDS solutions and incubate for 5 minutes at room temperature with gentle rocking.
  3. Wash sections in three changes of PBS , for 5 minutes each change, to eliminate remaining SDS solution. (It is critical to remove residual SDS as this will denature  antibodies, preventing effective binding.)
  4. Proceed with tissue blocking for immunohistochemistry.

References

  1. Brown D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, Alper S (1996)  Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS).Histochem Cell Biol. 105(4):261-7. PubMed Abstract
]]> Tris Buffer Antigen Retrieval Protocol https://www.protocolsonline.com/histology/immunohistochemistry-histology/tris-buffer-antigen-retrieval-protocol/ Tue, 24 Jul 2012 22:48:24 +0000 http://protocolsonline.com/?p=1410 Introduction

A Tris Buffer antigen retrieval solution is used with paraffin embedded, formalin or paraformaldehyde-fixed  tissues [1].  When tissues are fixed in cross-linking agents such as paraformaldehyde, these agents will covalently cross-link proteins, resulting a reduction in the available epitopes for antibody binding. Use of this basic antigen retrieval solution effectively unmasks some protein epitopes, but should be used with care as tissue may detach from slides somewhat more frequently than with other antigen retrieval solutions.

Materials

Tris Buffer ( 10 mM TBS, 0.05% Tween 20, pH 10.0)

  • Mix 1.21 g of Tris Base in 1000 ml of distilled water. Adjust the pH to 10.0 with 1 N NaOH and then add 0.5 ml of Tween 20.
  • Store at room temperature for up to 3 months; for extended storage, store at 4oC.

Procedure

  1. Deparaffinize and rehydrate tissue sections.
  2. Preheat a steam or a water bath containing a staining dish filled with  antigen retrieval buffer to 95-100oC.
  3. Immerse slides in the staining staining dish and incubate for 20-40 minutes; time is empirically determined with a suggestion for 20, 30 and 40 minutes as initial starting conditions.
  4. Remove the staining dish and allow slides to cool for 30 minutes before immersing in wash buffer in the next step. The cooling process prevents tissue from falling off the slide.
  5. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to eliminate remaining antigen retrieval solution.
  6. Proceed with tissue blocking for immunohistochemistry.

References

  1. Pileri SA, Roncador G, Ceccarelli C, et al (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol. 183(1):116-23. PubMed Abstract
]]> Tris Buffered Saline (TBS) Antigen Retrieval Protocol https://www.protocolsonline.com/histology/immunohistochemistry-histology/tris-buffered-saline-tbs-antigen-retrieval-protocol/ Tue, 24 Jul 2012 13:34:39 +0000 http://protocolsonline.com/?p=1403 Introduction

A Tris Buffered Saline (TBS) antigen retrieval solution is used with paraffin embedded, formalin or paraformaldehyde-fixed  tissues [1].  When tissues are fixed in cross-linking agents such as paraformaldehyde, these agents will covalently cross-link proteins, resulting a reduction in the available epitopes for antibody binding. Use of this basic antigen retrieval solution effectively unmasks some proteins epitopes.

Materials

Tris Buffered Saline (TBS) buffer ( 50 mM TBS, 0.05% Tween 20, pH 8.0)

  • Mix 6.1 g of Tris Base, 8.8 g of NaCl in 1000 ml of distilled water. Adjust the pH to 9.0 with concentrated HCl and then add 0.5 ml of Tween 20.
  • Store at room temperature for up to 3 months; for extended storage, store at 4oC.

Procedure

  1. Deparaffinize and rehydrate tissue sections.
  2. Preheat a steam or a water bath containing a staining dish filled with  antigen retrieval buffer to 95-100oC.
  3. Immerse slides in the staining staining dish and incubate for 20-40 minutes; time is empirically determined with a suggestion for 20, 30 and 40 minutes as initial starting conditions.
  4. Remove the staining dish and allow slides to cool for 30 minutes before immersing in wash buffer in the next step. The cooling process prevents tissue from falling off the slide.
  5. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to eliminate remaining antigen retrieval solution.
  6. Proceed with tissue blocking for immunohistochemistry.

References

  1. Pileri SA, Roncador G, Ceccarelli C, et al (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol. 183(1):116-23. PubMed Abstract
]]> Tris-EDTA Antigen Retrieval Protocol https://www.protocolsonline.com/histology/immunohistochemistry-histology/edta-tris-antigen-retrieval-protocol/ https://www.protocolsonline.com/histology/immunohistochemistry-histology/edta-tris-antigen-retrieval-protocol/#comments Tue, 24 Jul 2012 13:26:13 +0000 http://protocolsonline.com/?p=1394 Introduction

A Tris-EDTA buffer antigen retrieval solution is used with paraffin embedded, formalin or paraformaldehyde-fixed  tissues [1].  When tissues are fixed in cross-linking agents such as paraformaldehyde, these agents will covalently cross-linked proteins, resulting a reduction in the available epitopes for antibody binding. EDTA buffer is used to improve staining with low abundance epitopes and with antibodies that have weak affinity; it appears to enhance staining for many antibodies, although background staining is often increased.

Materials

Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 8.0)

  • Mix 1.21 g Tris Base, 0.37 g of EDTA in 1000 ml of distilled water. Adjust the pH to 9.0 with 1N sodium hydroxide and then add 0.5 ml of Tween 20.
  • Store at room temperature for up to 3 months; for extended storage, store at 4oC.

Procedure

  1. Deparaffinize and rehydrate tissue sections.
  2. Preheat a steam or a water bath containing a staining dish filled with  antigen retrieval buffer to 95-100oC.
  3. Immerse slides in the staining staining dish and incubate for 20-40 minutes; time is empirically determined with a suggestion for 20, 30 and 40 minutes as initial starting conditions.
  4. Remove the staining dish and allow slides to cool for 30 minutes before immersing in wash buffer in the next step. The cooling process prevents tissue from falling off the slide.
  5. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to eliminate remaining antigen retrieval solution.
  6. Proceed with tissue blocking for immunohistochemistry.

References

  1. Pileri SA, Roncador G, Ceccarelli C, et al (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol. 183(1):116-23. PubMed Abstract
]]> https://www.protocolsonline.com/histology/immunohistochemistry-histology/edta-tris-antigen-retrieval-protocol/feed/ 1 EDTA Buffer Antigen Retrieval Protocol https://www.protocolsonline.com/histology/immunohistochemistry-histology/edta-buffer-antigen-retrieval-protocol/ https://www.protocolsonline.com/histology/immunohistochemistry-histology/edta-buffer-antigen-retrieval-protocol/#comments Thu, 19 Jul 2012 12:43:56 +0000 http://protocolsonline.com/?p=1384 Introduction

An EDTA buffer antigen retrieval solution is used with paraffin embedded, formalin or paraformaldehyde-fixed  tissues[1-2].  When tissues are fixed in cross-linking agents such as paraformaldehyde, these agents will covalently cross-linked proteins, resulting a reduction in the available epitopes for antibody binding. EDTA buffer is used to improve staining with low abundance epitopes and with antibodies that have weak affinity; it appears to enhance staining for many antibodies, although background staining is often increased.

Materials

EDTA buffer (1 mM EDTA, 0.05% Tween 20, pH 8.0)

  • Mix 0.37 g of EDTA in 1000 ml of distilled water. Adjust the pH to 8.0 with 1N sodium hydroxide and then add 0.5 ml of Tween 20.
  • Store at room temperature for up to 3 months; for extended storage, store at 4oC.

Procedure

  1. Deparaffinize and rehydrate tissue sections.
  2. Preheat a steam or a water bath containing a staining dish filled with  antigen retrieval buffer to 95-100oC.
  3. Immerse slides in the staining staining dish and incubate for 20-40 minutes; time is empirically determined with a suggestion for 20, 30 and 40 minutes as initial starting conditions.
  4. Remove the staining dish and allow slides to cool for 30 minutes before immersing in wash buffer in the next step. The cooling process prevents tissue from falling off the slide.
  5. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to eliminate remaining antigen retrieval solution.
  6. Proceed with tissue blocking for immunohistochemistry.

References

  1. Pileri SA, Roncador G, Ceccarelli C, et al (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol. 183(1):116-23. PubMed Abstract
  2. Morgan JM, Navabi H, Schmid KW, Jasani B (1994) Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol. 174(4):301-7. PubMed Abstract
]]> https://www.protocolsonline.com/histology/immunohistochemistry-histology/edta-buffer-antigen-retrieval-protocol/feed/ 1 Citrate-EDTA Antigen Retrieval Protocol https://www.protocolsonline.com/histology/immunohistochemistry-histology/citrate-edta-antigen-retrieval-protocol/ https://www.protocolsonline.com/histology/immunohistochemistry-histology/citrate-edta-antigen-retrieval-protocol/#comments Thu, 19 Jul 2012 12:25:15 +0000 http://protocolsonline.com/?p=1382 Introduction

Citrate EDTA buffer antigen retrieval solution is used with paraffin embedded, formalin or paraformaldehyde-fixed  tissues[1-6]; the introduction of EDTA may, in some cases, improve the consistency of staining [7]. When tissues are fixed in cross-linking agents such as paraformaldehyde, these agents will covalently cross-linked proteins, resulting a reduction in the available epitopes for antibody binding. The result is weak or negligible levels of protein detection. Under elevated temperatures, citrate EDTA buffer has been shown to greatly improve detection of some proteins, although not all. Several antigen retrieval condition should be tested when optimizing stainng conditions.

Materials

 Citrate EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2)

  • Mix 1.92 g of anhydrous citric acid in 1000 ml of distilled water.
  • Add 0.74 g of EDTA.
  • Adjust the pH to 6.2 with 1N sodium hydroxide and then add 0.5 ml of Tween 20.
  • Store at room temperature for up to 3 months; for extended storage, store at 4oC.

Procedure

  1. Deparaffinize and rehydrate tissue sections.
  2. Preheat a steam or a water bath containing a staining dish filled with  antigen retrieval buffer to 95-100oC.
  3. Immerse slides in the staining staining dish and incubate for 20-40 minutes; time is empirically determined with a suggestion for 20, 30 and 40 minutes as initial starting conditions.
  4. Remove the staining dish and allow slides to cool for 30 minutes before immersing in wash buffer in the next step. The cooling process prevents tissue from falling off the slide.
  5. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to eliminate remaining antigen retrieval solution.
  6. Proceed with tissue blocking for immunohistochemistry.

References

  1. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 1993 Nov;41(11):1599-604. PubMed Abstract
  2. Kanai K, Nunoya T, Shibuya K, Nakamura T, Tajima M (1998) Variations in effectiveness of antigen retrieval pretreatments for diagnostic immunohistochemistry. Res Vet Sci. 64(1):57-61.PubMed Abstract
  3. Brown RW, Chirala R (1995) Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol. 8(5):515-20. PubMed Abstract
  4. Morgan JM, Navabi H, Schmid KW, Jasani B. Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol. 1994 Dec;174(4):301-7.PubMed Abstract
  5. Pellicer EM, Sundblad A (1994) Antigen retrieval by microwave oven with buffer of citric acid.Medicina (B Aires). 54(2):129-32.PubMed Abstract
  6. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 41(11):1599-604.PubMed Abstract
  7. 1. Sheriffs IN, Rampling D, Smith VV (2001) Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy. J Clin Pathol. 54(7):517-20. PubMed Abstract
]]> https://www.protocolsonline.com/histology/immunohistochemistry-histology/citrate-edta-antigen-retrieval-protocol/feed/ 1 Citrate Buffer Antigen Retrieval Protocol https://www.protocolsonline.com/histology/immunohistochemistry-histology/citrate-buffer-antigen-retrieval-protocol/ https://www.protocolsonline.com/histology/immunohistochemistry-histology/citrate-buffer-antigen-retrieval-protocol/#comments Wed, 18 Jul 2012 17:52:52 +0000 http://protocolsonline.com/?p=1374 Introduction

Citrate buffer antigen retrieval is used with paraffin embedded, formalin or paraformaldehyde-fixed  tissues[1-6]. When tissues are fixed in cross-linking agents such as paraformaldehyde, these agents will covalently cross-linked proteins, resulting a reduction in the available epitopes for antibody binding. The result is weak or negligible levels of protein detection. Under elevated temperatures, citrate buffer has been shown to greatly improve detection of some proteins, although not all. Several antigen retrieval condition should be tested when optimizing stainng conditions.

Materials

 Citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0)

  • Mix 1.92 g of anhydrous citric acid in 1000 ml of distilled water. Adjust the pH to 6.0 with 1N sodium hydroxide and then add 0.5 ml of Tween 20.
  • Store at room temperature for up to 3 months; for extended storage, store at 4oC.

Sodium Citrate buffer (10 mM sodium citrate acid, 0.05% Tween 20, pH 6.0)

  • Mix 2.94 g of dihydrate tri-sodium cirate in 1000 ml of distilled water. Adjust the pH to 6.0 with 1N sodium hydroxide and then add 0.5 ml of Tween 20.
  • Store at room temperature for up to 3 months; for extended storage, store at 4oC.

Procedure

  1. Deparaffinize and rehydrate tissue sections.
  2. Preheat a steam or a water bath containing a staining dish filled with  antigen retrieval buffer to 95-100oC. Either citrate or sodium citrate solution can be used;  sodium citrate is often the buffer of choice.
  3. Immerse slides in the staining staining dish and incubate for 20-40 minutes; time is empirically determined with a suggestion for 20, 30 and 40 minutes as initial starting conditions.
  4. Remove the staining dish and allow slides to cool for 30 minutes before immersing in wash buffer in the next step. The cooling process prevents tissue from falling off the slide.
  5. Rinse sections in two changes of a buffered solution, such as PBS or TBS, in order to eliminate remaining antigen retrieval solution and to neutralize acidity.
  6. Proceed with tissue blocking for immunohistochemistry.

References

  1. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 1993 Nov;41(11):1599-604. PubMed Abstract
  2. Kanai K, Nunoya T, Shibuya K, Nakamura T, Tajima M (1998) Variations in effectiveness of antigen retrieval pretreatments for diagnostic immunohistochemistry. Res Vet Sci. 64(1):57-61.PubMed Abstract
  3. Brown RW, Chirala R (1995) Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol. 8(5):515-20. PubMed Abstract
  4. Morgan JM, Navabi H, Schmid KW, Jasani B. Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol. 1994 Dec;174(4):301-7.PubMed Abstract
  5. Pellicer EM, Sundblad A (1994) Antigen retrieval by microwave oven with buffer of citric acid.Medicina (B Aires). 54(2):129-32.PubMed Abstract
  6. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 41(11):1599-604.PubMed Abstract
]]> https://www.protocolsonline.com/histology/immunohistochemistry-histology/citrate-buffer-antigen-retrieval-protocol/feed/ 1