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7TM Immunohistochemistry Protocol

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Note: This protocol is designed for the staining of formalin-fixed, paraffin-embedded (FFPE) tissue sections.

1. Buffers and Reagents

Use double distilled water for buffer preparation or water with the same grade of purity.

  • Citrate buffer: C6H8O7 x H2O: 1.8 mM, C6H5Na3O7 x H2O: 8.7 mM, pH 6.0
  • PBS: Phosphate Buffered Saline (NaCl: 150 mM, KH2PO4: 50 mM)
  • PBS/BSA: PBS with 1% BSA (bovine serum albumin)

2. Deparaffinization, Rehydration and Antigen Unmasking

  1. Wash sections with xylene for 20 min. Aspirate xylene. Repeat 3-times.
  2. Wash sections with 100% ethanol for 20 min. Aspirate ethanol. Repeat 3-times.
  3. Blocking of endogenous peroxidase: Incubation with 0.15% H2O2 in methanol for 45 min.
  4. Wash sections with 95% ethanol for 2 min. Aspirate ethanol. Repeat 2-times.
  5. Wash sections with 80% ethanol for 2 min. Aspirate ethanol.
  6. Wash sections with 70% ethanol for 2 min. Aspirate ethanol.
  7. Wash sections with water for 5 min. Aspirate water. Repeat 2-times.
  8. Use a microwave for the following boiling/cooling cycles in citrate buffer:
  • boil for 8 min
  • cool at room temperature for 4 min
  • boil for 4 min
  • cool at room temperature for 4 min
  • boil for 4 min
  • cool at room temperature for 20 min
  • aspirate citrate buffer and incubate for 5 min in PBS/BSA.

3. Staining and Mounting

  1. We recommend the use of a Shandon™ Sequenza™ staining rack for the following steps.
  2. Wash with 1.5 ml PBS/BSA for 5 min.
  3. Incubate sections with 300 µl 7TM Premium IHC-Grade Antibodies at a dilution of 1:100 in PBS/BSA for 1-2 hours at room temperature or at 4 °C overnight (we recommend the incubation overnight).
  4. Wash with 1.5 ml PBS/BSA for 5 min.
  5. Incubate sections with 150 µl biotinylated anti-rabbit-IgG of your choice for 20 min.
  6. Wash with 1.5 ml PBS/BSA for 5 min.
  7. Incubate sections with 150 µl peroxidase-conjugated streptavidin of your choice for 20 min.
  8. Wash with 1.5 ml PBS/BSA for 5 min.
  9. Incubate in 3-amino-9-ethylcarbazole (AEC) solution of your choice for 15 min. Repeat 2-times.
  10. Wash with 1.5 ml water for 5 min.
  11. Wash with water for 1 min. Repeat 2-times.
  12. Incubate sections with hematoxylin solution of your choice for 5 min.
  13. Wash with water for 2.5 min
  14. Dip sections in 250 ml water containing 1.4 ml concentrated ammonia. Repeat 5-times.
  15. Wash with water for 2.5 min.
  16. Mount sections by using an aqueous mounting media on microscope slides.

For more information please contact us:

E-Mail:       support@7tmantibodies.com
Fon:           0049-151-20130575
FAX:           0049-3641-2414958

Note: This protocol is designed for the staining of formalin-fixed, paraffin-embedded (FFPE) tissue sections. 1. Buffers and Reagents Use double distilled water for buffer preparation or... read more »
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7TM Immunohistochemistry Protocol

Note: This protocol is designed for the staining of formalin-fixed, paraffin-embedded (FFPE) tissue sections.

1. Buffers and Reagents

Use double distilled water for buffer preparation or water with the same grade of purity.

  • Citrate buffer: C6H8O7 x H2O: 1.8 mM, C6H5Na3O7 x H2O: 8.7 mM, pH 6.0
  • PBS: Phosphate Buffered Saline (NaCl: 150 mM, KH2PO4: 50 mM)
  • PBS/BSA: PBS with 1% BSA (bovine serum albumin)

2. Deparaffinization, Rehydration and Antigen Unmasking

  1. Wash sections with xylene for 20 min. Aspirate xylene. Repeat 3-times.
  2. Wash sections with 100% ethanol for 20 min. Aspirate ethanol. Repeat 3-times.
  3. Blocking of endogenous peroxidase: Incubation with 0.15% H2O2 in methanol for 45 min.
  4. Wash sections with 95% ethanol for 2 min. Aspirate ethanol. Repeat 2-times.
  5. Wash sections with 80% ethanol for 2 min. Aspirate ethanol.
  6. Wash sections with 70% ethanol for 2 min. Aspirate ethanol.
  7. Wash sections with water for 5 min. Aspirate water. Repeat 2-times.
  8. Use a microwave for the following boiling/cooling cycles in citrate buffer:
  • boil for 8 min
  • cool at room temperature for 4 min
  • boil for 4 min
  • cool at room temperature for 4 min
  • boil for 4 min
  • cool at room temperature for 20 min
  • aspirate citrate buffer and incubate for 5 min in PBS/BSA.

3. Staining and Mounting

  1. We recommend the use of a Shandon™ Sequenza™ staining rack for the following steps.
  2. Wash with 1.5 ml PBS/BSA for 5 min.
  3. Incubate sections with 300 µl 7TM Premium IHC-Grade Antibodies at a dilution of 1:100 in PBS/BSA for 1-2 hours at room temperature or at 4 °C overnight (we recommend the incubation overnight).
  4. Wash with 1.5 ml PBS/BSA for 5 min.
  5. Incubate sections with 150 µl biotinylated anti-rabbit-IgG of your choice for 20 min.
  6. Wash with 1.5 ml PBS/BSA for 5 min.
  7. Incubate sections with 150 µl peroxidase-conjugated streptavidin of your choice for 20 min.
  8. Wash with 1.5 ml PBS/BSA for 5 min.
  9. Incubate in 3-amino-9-ethylcarbazole (AEC) solution of your choice for 15 min. Repeat 2-times.
  10. Wash with 1.5 ml water for 5 min.
  11. Wash with water for 1 min. Repeat 2-times.
  12. Incubate sections with hematoxylin solution of your choice for 5 min.
  13. Wash with water for 2.5 min
  14. Dip sections in 250 ml water containing 1.4 ml concentrated ammonia. Repeat 5-times.
  15. Wash with water for 2.5 min.
  16. Mount sections by using an aqueous mounting media on microscope slides.

For more information please contact us:

E-Mail:       support@7tmantibodies.com
Fon:           0049-151-20130575
FAX:           0049-3641-2414958

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