Histology

Mtt Assay Using Hl60 Cells

Plate 20,000 cells/well add extract

incubate for 3 days

Add 50 ul MTT

Incubate for 4 hrs

Centrifuge 3500 g for 10 mins

Discard medium ,add 50 ul DMSO.

Shake 5 mins

Protocol For Chromogenic Staining Of Cryostat Sections

REAGENTS REQUIRED

  • Wash Buffer: 1X PBS??(0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4)
  • Incubation Buffer:??1% bovine serum albumin, 1% normal donkey serum, 0.3% Triton?? X-100, and 0.01% sodium azide in PBS
  • Primary Antibodies
  • Cell and Tissue Staining Kits:??Kits include Biotinylated Secondary Antibodies, Serum Blocking Reagent, Peroxidase Blocking Reagent, Avidin Blocking Reagent, Biotin Blocking Reagent, HRP-Streptavidin Conjugate (HSS-HRP), and Chromogen Solution. Kits are available with chromogenic substrates 3,3??? Diaminobenzidine (DAB, brown precipitate) or 3-amino-9-ethylcarbazole (AEC, red precipitate).
  • DAB Enhancer??(Catalog #??CTS010)
  • Hematoxylin
  • Aqueous Mounting Medium??(Catalog #??CTS011)
  • Antigen Retrieval Reagents??(if required;??Protocol for Heat-induced Epitope Retrieval (HEIR))

MATERIALS

PROCEDURE

This staining protocol has been developed and optimized for use with R&D Systems??Cell and Tissue Staining Kits.

  1. When staining cryostat sections stored in a freezer, thaw the slides at room temperature for 10-20 minutes.
  2. Rehydrate the slides in wash buffer for 10 minutes. Drain the excess wash buffer.
    Note: Excessive fixation may result in the masking of an epitope and strong non-specific background signal that can obscure specific labeling. If necessary, an??antigen retrieval protocol??can be performed at this time. However, many antigen retrieval techniques are too harsh for cryostat cut tissue sections.
    Note: Endogenous peroxidase and biotin can react with secondary reagents and cause non-specific background staining. R&D Systems??Cell and Tissue Staining Kits??contain reagents to address these potential artifacts (applied in protocol steps 4-8).
  3. Surround the tissue with a hydrophobic barrier using a barrier pen.
  4. To quench endogenous peroxidase activity, incubate the sample with 1-3 drops peroxidase blocking reagent (3% H2O2??in water or methanol) for 5-15 minutes.
  5. Rinse the sample, and gently wash in wash buffer for 5 minutes.
  6. To reduce non-specific hydrophobic interactions between the primary antibodies and the tissue, block the section with 1-3 drops of serum blocking reagent for 15 minutes. Drain the slides, and wipe away any excess blocking reagent before proceeding to the next step. Do not rinse.
  7. To block binding to endogenous biotin, incubate the sample with 1-3 drops of avidin blocking reagent for 15 minutes. Rinse the sample with wash buffer, drain slides, and wipe away any excess wash buffer.
  8. To block subsequent binding to the avidin applied in step 6, incubate the sample with 1-3 drops of biotin blocking reagent for 15 minutes. Rinse with wash buffer, drain the slides, and wipe away any excess wash buffer.
  9. Incubate the sample with primary antibodies in Incubation Buffer. Follow manufacturer???s recommendations regarding working dilution for the primary antibody. For chromogenic IHC staining of frozen tissue sections using R&D Systems antibodies, it is recommended to incubate overnight at 2-8 ??C. This incubation regime allows for optimal specific binding of antibodies to tissue targets and reduces non-specific background staining. These variables may need to be optimized for your system
    Note:??Appropriate controls??are critical for the accurate interpretation of IHC/ICC results. All IHC/ICC experiments should include a negative control using the incubation buffer with no primary antibody to identify non-specific staining of the secondary reagents. Additional controls can be employed to support the specificity of staining generated by the primary antibody. These include absorption controls, isotype matched controls (for monoclonal primary antibodies), and tissue type controls.
  10. Rinse the sample with wash buffer. Wash 3 times with wash buffer for 5 minutes each, and drain the slides.
  11. Incubate the sample with 1-3 drops of biotinylated secondary antibodies for 30-60 minutes, adjusting the incubation time depending on the thickness of the section (approximately 30 minutes for 5-10 ??m thick sections and 60 minutes for 10-20 ??m thick sections).
  12. Rinse with wash buffer 3 times for 15 minutes each and drain the slides.
  13. Incubate the sample with 1-3 drops of High Sensitivity Streptavidin-HRP conjugate (HSS-HRP) for 30 minutes. This signal amplification technique is referred to as the labeled streptavidin-biotin (LSAB) method.
    Note: High Sensitivity Streptavidin is a chemical analog of Streptavidin that has little net positive charge at neutral or slightly alkaline pH and will interact only with biotin attached to secondary antibodies. HSS-HRP shows little or no non-specific binding to phospholipids, nucleic acids, and carbohydrate binding proteins.
  14. Rinse and wash 3 times in wash buffer for 2 minutes each.
  15. Calculate the working volume of DAB/AEC Chromogen Solution given that 100-200 ??L is required to cover the entire tissue section on a slide. Add 1-5 drops of DAB/AEC Chromogen Solution to cover the entire tissue section, and incubate for 3-20 minutes. Monitor intensity of the tissue staining under a microscope. Colored precipitate will localize to the sites of antigen expression as the chromogenic substrate is converted by HRP enzyme into insoluble end product.
    Note: DAB and AEC are hazardous materials. Gloves and safety glasses should be worn and all steps performed inside a fume hood. Please refer to the MSDS for safe deactivation.
    Note: If required, DAB Enhancer (Catalog #??CTS010) can be used to intensify the DAB Chromogen solution.
  16. Rinse the sample with wash buffer 3 times for 10 minutes each.
  17. Rinse in deionized H2O and drain the slides.
  18. Stained tissue can be mounted either without nuclear counterstaining or counterstained with nuclear counterstain hematoxylin for better visualization of the tissue morphology.
    Note: Hematoxylin counterstain can obscure visualization of targets localized in cell nuclei.
  19. Cover stained tissue with a coverslip of an appropriate size, place slides vertically on a filter paper or towel to drain excess mounting medium and allow them to dry.
    Note: Unlike DAB, AEC is soluble in alcohols and xylene. Tissue sections subjected to an HRP-AEC protocol should be coverslipped using only aqueous mounting media.
  20. Visualize staining of tissue under a microscope using a bright-field illumination.
    Note: Initial IHC/ICC studies often require further optimization and/or additional

Protocol For Cryopreservation Of Tissues Prior To Fixation

This method utilizes frozen tissues that are fixed after snap-freezing and sectioning with a cryostat.

PROCEDURE

  1. Immediately snap freeze fresh tissue in isopentane mixed with dry ice, and keep at -70 ??C. Do not allow frozen tissue to thaw before cutting.
  2. Embed the tissue completely in OCT compound prior to cryostat sectioning.
  3. Cut cryostat sections at 5-10 ??m and mount on gelatin-coated histological slides.
    Note: The suggested cryostat temperature is between -15 and -23 ??C. The section will curl if the specimen is too cold. If it is too warm, it will stick to the knife.
  4. Air dry the sections for 30 minutes at room temperature to prevent sections from falling off the slides during antibody incubations.
    Note: Slides can be stored unfixed for up to one year at -80 ??C. Frozen tissue samples saved for later analysis should be stored intact.
  5. Immediately add 50 ??L of ice-cold Fixation Buffer to each tissue section upon removal from the freezer.
  6. Fix for 8 minutes at 2-8 ??C or, optimally, at -20 ??C for 20 minutes.

Protocol For Fixing And Sectioning Frozen Tissues

REAGENTS REQUIRED

  • Formaldehyde Fixative Solution:??85 mM Na2HPO4, 75 mM KH2P04, 4% paraformaldehyde, and 14% (v/v) saturated picric acid, pH 6.9. (Picric acid is optional, enhances preservation of morphology in some tissues).
  • Sucrose Solution:??130 mM Na2HPO4, 30 mM KH2P04, 10% (w/v) sucrose, 0.01% sodium azide and 0.03% Bacitracin, pH 7.2
  • OCT Embedding Compound
  • Isopentane
  • Dry Ice

PROCEDURE

This technique utilizes formaldehyde-based fixation before the tissue is frozen and sectioned using a cryostat.

  1. To preserve tissue morphology and retain the antigenicity of the target molecules, fix the tissue by vascular perfusion with 500 – 700 mL of Formaldehyde Fixative Solution.
  2. Perfuse the animal with 400 mL of Sucrose Solution. This step cryoprotects tissues frozen for cutting on a cryostat.
    Note: When it is not possible to fix by perfusion, dissected tissue may be fixed by immersion in a 10% formalin solution for 4-8 hours at room temperature. It is commonly accepted that the volume of fixative should be 50 times greater than the size of the immersed tissue. Avoid fixing the tissue for greater than 24 hours since tissue antigens may either be masked or destroyed.
    Note: R&D Systems scientists perfuse fix all rodent tissue with the exception of lung, spleen, and embryonic tissue, which are immersion fixed.
  3. Dissect the tissue, mount in OCT embedding compound, and freeze at -20 to -80 ??C.
  4. Cut 5-15 ??m thick tissue sections using a cryostat.
    Note: The suggested cryostat temperature is between -15 and -23 ??C. The section will curl if the specimen is too cold. If it is too warm, it will stick to the knife.
  5. Thaw-mount the sections onto gelatin-coated histological slides. Slides are pre-coated with gelatin to enhance adhesion of the tissue. Please refer to the??Protocol for the Preparation of Gelatin-coated Slides for Histological Tissue Sections??for instructions on how to prepare gelatin-coated slides.
  6. Dry the slides for 30 minutes on a slide warmer at 37 ??C. Slides containing cryostat sections can be stored at -20 to -70 ??C for up to 12 months.

Protocol For Chromogenic Staining Of Paraffinembedded Sections

REAGENTS REQUIRED

  • Wash Buffer: 1X PBS??(0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4)
  • Incubation Buffer:??1% bovine serum albumin, 1% normal donkey serum, 0.3% Triton?? X-100, and 0.01% sodium azide in PBS
  • Primary Antibodies
  • Cell and Tissue Staining Kits:??Kits include Biotinylated Secondary Antibodies, Serum Blocking Reagent, Peroxidase Blocking Reagent, Avidin Blocking Reagent, Biotin Blocking Reagent, High Sensitivity Streptavidin-HRP Conjugate (HSS-HRP), and Chromogen Solution. Kits are available with chromogenic substrates 3,3??? Diaminobenzidine (DAB, brown precipitate) or 3-amino-9-ethylcarbazole (AEC, red precipitate).
  • DAB Enhancer??(Catalog #??CTS010)
  • Hematoxylin Counterstain
  • Aqueous Mounting Medium??(Catalog #??CTS011)
  • Antigen Retrieval Reagents??(if required;??Protocol for Heat-induced Epitope Retrieval (HEIR))

MATERIALS

PROCEDURE

This staining protocol has been developed and optimized for use with R&D Systems??Cell and Tissue Staining Kits.

  1. Tissue must be rehydrated before commencing staining protocol.
    1. Immerse the slides in xylene (mixed isomers) 2 times for 10 minutes each.
    2. Immerse the slides in 100% alcohol 2 times for 10 minutes each.
    3. Immerse the slides in 95% alcohol for 5 minutes.
    4. Immerse the slides in 70% alcohol for 5 minutes.
    5. Immerse the slides in 50% alcohol for 5 minutes.
    6. Rinse the slides with deionized H2O.
    7. Rehydrate the slides with wash buffer for 10 minutes. Drain the excess wash buffer.

    Note: Excessive fixation may result in the masking of an epitope and strong non-specific background signal that can obscure specific labeling. If necessary, an??antigen retrieval protocol??can be performed at this time.

    Note: Endogenous peroxidase and biotin can react with secondary reagents and cause non-specific background staining. R&D Systems??Cell and Tissue Staining Kits??contain reagents to address these potential artifacts (applied in protocol steps 3 ??? 7).

  2. Surround the tissue with a hydrophobic barrier using a barrier pen.
  3. To quench endogenous peroxidase activity, incubate the sample with 1-3 drops peroxidase blocking reagent (3% H2O2??in water or methanol) for 5-15 minutes.
  4. Rinse the sample, then gently wash in wash buffer for 5 minutes.
  5. To reduce non-specific hydrophobic interactions between the primary antibodies and the tissue, block the section with 1-3 drops of serum blocking reagent for 15 minutes. Drain the slides and wipe away any excess blocking reagent before proceeding to the next step. Do not rinse.
  6. To block binding to endogenous biotin, incubate the sample with 1-3 drops of avidin blocking reagent for 15 minutes. Rinse the sample with wash buffer, drain slides, and wipe away any excess wash buffer.
  7. To block subsequent binding to the avidin applied in step 6, incubate the sample with 1-3 drops of biotin blocking reagent for 15 minutes. Rinse with wash buffer, drain the slides, and wipe away any excess wash buffer.
  8. Incubate the sample with primary antibodies in Incubation Buffer. Follow manufacturer???s recommendations regarding working dilution for the primary antibody. For chromogenic IHC staining of paraffin-embedded tissue sections using R&D Systems antibodies, it is recommended to incubate overnight at 2-8 ??C. This incubation regime allows for optimal specific binding of antibodies to tissue targets and reduces non-specific background staining. These variables may need to be optimized for your system.
    Note:??Appropriate controls??are critical for the accurate interpretation of IHC/ICC results. All IHC/ICC experiments should include a negative control using the incubation buffer with no primary antibody to identify non-specific staining of the secondary reagents. Additional controls can be employed to support the specificity of staining generated by the primary antibody. These include absorption controls, isotype matched controls (for monoclonal primary antibodies), and tissue type controls.
  9. Rinse the sample with wash buffer. Wash 3 times with wash buffer for 5 minutes each, and drain the slides.
  10. Incubate the sample with 1-3 drops of biotinylated secondary antibodies for 30-60 minutes, adjusting the incubation time depending on the thickness of the section (approximately 30 minutes for 5-10 ??m thick sections and 60 minutes for 10-20 ??m thick sections).
  11. Rinse with wash buffer 3 times for 15 minutes each and drain the slides.
  12. Incubate the sample with 1-3 drops of High Sensitivity Streptavidin-HRP conjugate (HSS-HRP) for 30 minutes. This signal amplification technique is referred to as the labeled streptavidin-biotin (LSAB) method.
    Note: High Sensitivity Streptavidin is a chemical analog of Streptavidin that has little net positive charge at neutral or slightly alkaline pH and will interact only with biotin attached to secondary antibodies. HSS-HRP shows little or no non-specific binding to phospholipids, nucleic acids, and carbohydrate binding proteins.
  13. Rinse and wash 3 times in wash buffer for 2 minutes each.
  14. Calculate the required working volume of DAB/AEC Chromogen Solution given that 100-200 ??L is required to cover the entire tissue section on a single slide. Add 1-5 drops of DAB/AEC Chromogen Solution to cover the entire tissue section and incubate for 3-20 minutes. Monitor the intensity of the tissue staining under a light microscope. Colored precipitate will localize to the sites of antigen expression as the chromogenic substrate is converted by HRP enzyme into insoluble end product.
    Note: DAB and AEC are hazardous materials. Gloves and safety glasses should be worn and all steps performed inside a fume hood. Please refer to the MSDS for safe deactivation.
    Note: If required, DAB Enhancer (Catalog #??CTS010) can be used to intensify the DAB Chromogen solution.
  15. Rinse the sample with wash buffer 3 times for 10 minutes each.
  16. Rinse in deionized H2O and drain the slides.
  17. Stained tissue can be mounted either without nuclear counterstaining or counterstained with nuclear counterstain hematoxylin for better visualization of the tissue morphology.
    Note: Hematoxylin counterstain can obscure visualization of targets localized in cell nuclei.
  18. Cover stained tissue with a coverslip of an appropriate size, place slides vertically on filter paper or a towel to drain excess mounting medium, and allow them to dry.
    Note: Unlike DAB, AEC is soluble in alcohols and xylene. Tissue sections subjected to an HRP-AEC protocol should be coverslipped using only aqueous mounting media.
  19. Visualize staining of tissue under a microscope using a bright-field illumination.
    Note: Initial IHC/ICC studies often require further optimization and/or additional

Protocol For Fixing And Sectioning Paraffinembedded Tissues

REAGENTS REQUIRED

  • Formaldehyde Fixative Solution:??85 mM Na2HPO4, 75 mM KH2P04, 4% paraformaldehyde, and 14% (v/v) saturated picric acid, pH 6.9. (Picric acid is optional, enhances preservation of morphology in some tissues)
  • Wash Buffer: 1X PBS??(0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4)
  • Ethanol:??100%, 90%, 70%
  • Xylene??(mixed isomers)
  • Paraffin

PROCEDURE

  1. To preserve tissue morphology and retain the antigenicity of the target molecules, fix the tissue by vascular perfusion with 500 – 700 mL of Formaldehyde Fixative Solution.
    Note: When it is not possible to fix by perfusion, dissected tissue may be fixed by immersion in a 10% formalin solution for 4-8 hours at room temperature. It is commonly accepted that the volume of fixative should be 50 times greater than the size of the immersed tissue. Avoid fixing the tissue for greater than 24 hours since tissue antigens may either be masked or destroyed.
    Note: R&D Systems scientists perfuse fix all rodent tissue with the exception of lung, spleen, and embryonic tissue, which are immersion fixed.
  2. Because paraffin is immiscible with water, tissue must be dehydrated before adding molten paraffin wax.
    1. Immerse the tissue in 70% ethanol three times for 30 minutes each at room temperature.
    2. Immerse the tissue in 90% ethanol two times for 30 minutes each at room temperature.
    3. Immerse the tissue in 100% ethanol three times for 30 minutes each at room temperature.
    4. Immerse the tissue in xylene (mixed isomers) three times for 20 minutes each at room temperature.
  3. Embed the tissue in paraffin at 58 ??C. Tissues can be embedded into paraffin using specialized automated tissue processing systems.
  4. Cut 5 – 15 ??m thick tissue sections using a rotary microtome.
  5. Float the sections in a 56 ??C water bath.
  6. Mount the sections onto gelatin-coated histological slides. Slides are pre-coated with gelatin to enhance adhesion of the tissue. Please refer to the??Protocol for the Preparation of Gelatin-coated Slides for Histological Tissue Sections??for instructions on how to prepare gelatin-coated slides.
  7. Dry the slides overnight at room temperature. Slides with paraffin-embedded sections can be stored either at room temperature or in at 2-8 ??C for several years in slide storage boxes.

Diffquick Ii Staining Protocol For Helicobacter Pylori

Principle:??Helicobacter (formerly Campylobacter) pylori is a large curved gram negative bacillus that often colonizes the mucosal surface of the stomach. The Diff-Quik II stain, similar in action to the Giemsa stain, stains the bacillus fairly sensitively, though not at all specifically.

??

Specimen:??The usual specimen is a gastric or duodenal biopsy specimen, fixed in 10% formalin or neutral buffered formalin, paraffin processed, and cut in the usual fashion. Diff-Quik II can also be used as a general bacterial stain for other tissues.

??

Reagents:??Diff-Quik II??, a proprietary mixture of the thiazine dyes methylene blue and azure A in a water solution with a buffer, is manufactured by American Scientific Products, and the name is a registered trademark. Generic equivalents of this stain exist, such as??Hema-Diff Stain Kit??can be purchased from IHC World at??IHC Store. Store at room temperature. Dispose of it down the drain, with plenty of water.

??

Procedure:

??

1. Deparaffinize sections and hydrate to water.

2. Stain 2 to 3 minutes in Diff-Quik II stain in a Coplin jar.

3. Rinse rapidly in water.

4. Dehydrate rapidly in 100% alcohol and pass to xylene or xylene substitute.

5. Mount in resin.

??

Control:

??

Run a control with every stain run. The control may be a gastric biopsy specimen known to be positive, or a specially prepared control (see reference below).

??

Result:

??

Bacteria and fungi are stained dark blue, with very distinct morphologic features. Cell nuclei and other histological structures are also distinctly blue. Helicobacter pylori is seen as large curved, helical, or gull-wing shaped bacilli. Gastrospirillum hominis is clearly stained and easily identified, in the author’s experience of a single case.

Diffquick Diffquik Staining Protocol

Principle

“Diff-Quick” is a proprietory brand of a Romanowski stain. The Romanowski group of stains are defined as being the black precipitate formed from the addition of aqueous solutions of methylene blue and eosin, dissolved in methanol.?? The variants of the Romanowski group differ in the degree of oxidation (polychroming) of the methylene blue stain prior to the precipitation.

??

The stain class was originally designed to incorporate cytoplasmic (pink) staining with nuclear (blue) staining and fixation as a single step for smears and thin films of tissue (spread preparations of omentum).?? Minor modifications of working stain concentration and staining time have been made over the years for fixed tissue sections.

??

Technical Points

??

(step 5) – exposure to alcohol should be as brief as possible to prevent excessive decolourisation.

??

Method

??

Paraffin Sections

??

1.????????????Bring sections to distilled water

2.????????????Stain with??“Diff Quick” solution II??.30 secs

3.????????????Counterstain (optional) with “Diff Quick” solution??I for??30 seconds

4.????????????Rinse rapidly in tap water

5.????????????Rapidly dehydrate in absolute alcohol

6.????????????Clear and mount

??

Smears/Imprints

??

1.????????????Air-dry the smear

2.????????????Fix in “Diff Quick” Fixative (or methanol)??for??30 secs/drain

3.????????????Stain with??“Diff Quick” solution II for??30 secs/drain

4.????????????Counterstain (optional) with “Diff Quick” solution I??for??30 secs/drain

5.????????????Rinse in tap water to remove excess stain

6.????????????Rapidly dehydrate in absolute alcohol

7.????????????Clear and mount

??

Results

??

helicobacter………………………………….dark blue

background…………………………………..light blue

platelets……………………………………..violet to purple

??

neutrophils??????nucleus?????????………???????????????dark blue

???????????????????????????????????? cytoplasm….????????????????????????..pale pink

eosinophils??????nucleus……..????????????????????????.blue

???????????????????????????????????? cytoplasm…..????????????????????????.blue

???????????????????????????????????? granules……………………red to red/orange

basophils?????????? nucleus………?????????????????????.purple or dark blue

???????????????????????????????????? granules……..??????????????? ??????dark purple/black

monocytes?????? nucleus(lobated)???????????? ??????violet

???????????????????????????????????? cytoplasm……..??????…??????..sky blue??????????????????????

Modified High Ph Congo Red Staining Protocol For Amyloid

Description:??This??high pH??Congo Red stain??is used for the detection of amyloid on formalin-fixed, paraffin-embedded tissue sections with amyloidosis, and may be used for frozen sections as well. The amyloid deposits will be stained red and the nuclei will be stained blue.??The thickness of sections is usually 5 um. But in case of inadequate amyloid deposits, 10um thick sections will be more satisfactory.??Congo red stains amyloid in tissue sections.

??

Fixation:??10% formalin.

??

Section:??paraffin sections at 5 um (in case inadequate amyloid deposits, use 10 um thick sections).

??

Solutions and Reagents:

??

Congo Red Stock Solution:

??

????????????Congo red (Sigma, Cat# C-6277) ——— 0.3 g

????????????Sodium chloride —————————- 0.3 g

????????????80% Alcohol ——————————— 100 ml

??????

1% Sodium Hydroxide:

??

????????????Sodium hydroxide ————————- 1 g

????????????Distilled water —————————– 100 ml

??

Congo Red Working Solution:

??

??????????1% Sodium hydroxide ———————- 0.5 ml

??????????Congo red stock solution —————— 50 ml

??????????Mix well, filter and use within 20 minutes.

??

Alkaline Alcohol Solution:

??

??????????1% Sodium hydroxide?? ——————— 1 ml

????????????50% alcohol ——————————– 100 ml

??

Procedure:

??

1.??????Deparaffinize and hydrate sections to distilled water.

2.??????Stain in congo red working solution for 10 minutes.

3.??????Rinse in distilled water.????

4.??????Differentiate quickly (5-10 dips) in alkaline alcohol solution.

5.??????Rinse in tap water.

6.??????Counterstain in Gill’s hematoxylin for 30 seconds.

7.??????Rinse in tap water for 2 minutes.

8.??????Dip in ammonia water (add a few drops of ammonium hydroxide to tap water and mix well) for 30 seconds or until sections turn blue.

9.??????Rinse in tap water for 5 minutes.

10.??Dehydrate through 95% alcohol, 100% alcohol

11.??Clear in xylene and mount with resinous mounting medium.

??

Results:

??

????????????Amyloid, elastic tissue, eosinophil granules ——- red

????????????Nuclei ————————————————– blue????

Bennholds Congo Red Staining Protocol For Amyloid

Description:??This Bennhold’s Congo Red stain??is used for the detection of amyloid on formalin-fixed, paraffin-embedded tissue sections with amyloidosis, and may be used for frozen sections as well. The amyloid deposits will be stained red and the nuclei will be stained blue.??The thickness of sections is usually 5 um. But in case of inadequate amyloid deposits, 10um thick sections will be more satisfactory.??Congo red stains amyloid in tissue sections.

??

Fixation:??10% formalin.

??

Section:??paraffin sections at 5 um (in case inadequate amyloid deposits, use 10 um thick sections).

??

Solutions and Reagents:????

??

1% Congo Red Solution:

??

????????????Congo red (Sigma, Cat# C-6277) —— 1 g

????????????Distilled water ————————— 100 ml

?? ??

1% Sodium Hydroxide:

??

????????????Sodium hydroxide ———————— 1 g

????????????Distilled water —————————- 100 ml

??

Alkaline Alcohol Solution:

??

????????????1% Sodium hydroxide?? ——————– 1 ml

????????????50% alcohol ——————————– 100 ml

??

Procedure:

??

1.??????Deparaffinize and hydrate sections to distilled water.

2.??????Stain in Congo red solution for 30-60 minutes.

3.??????Rinse in distilled water. ??

4.??????Differentiate rapidly (5-10 dips) in alkaline alcohol solution.

5.??????Rinse in running tap water for 5 minutes.

6.??????Counterstain in Gill’s hematoxylin for 30 seconds.

7.??????Rinse in tap water for 1 minute.

8.??????Dip in ammonia water (add a few drops of ammonium hydroxide to tap water and mix well) for 30 seconds or until sections turn blue.

9.??????Rinse in tap water for 5 minutes.

10.??Dehydrate through 95% alcohol, 100% alcohol

11.??Clear in xylene and mount with resinous mounting medium.

??

Results:

??

????????????Amyloid, elastic fibers, eosinophil granules ————- red

????????????Nuclei ——————————————————– blue????