ICAM-1的表达
CD54 (ICAM-1) Immunohistochemical Staining Protocol
Description: The intercellular adhesion molecule-1 (ICAM-1), also referred to as CD54, is an integral membrane protein of the immunoglobulin superfamily and plays a role in the cell adhesion. CD54 is found on resting (weak) and activated (moderate) lymphocytes and monocytes.
Fixation: Formalin-fixed, paraffin embedded sections and acetone fixed frozen sections.
Positive Controls: Tonsil, liver, kidney.
Solutions and Reagents:
Primary Antibody:
Mouse Anti-Rat CD54 (ICAM-1) (Pharmingen, Cat# 550302). Optimal dilution 1:50. Species Reactivity: Rat, mouse (refer to antibody datasheet for more information).
Secondary Antibody:
Horse Anti-Mouse IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-2000). Optimal dilution 1:500.
Detection Reagent:
HRP-Streptavidin (Vector Laboratories, Cat# SA-5004). Optimal dilution 1:500
Procedure:
1. Paraffin sections to distilled water.
2. Epitope Retrieval: Use Proteinase K Epitope Retrieval Method. Briefly, incubate sections with proteinase K solution in humidified chamber for 20 minutes at 37 °C (incubation time may need to be adjusted depending on degree of fixation and type of tissues). Allow sections to cool at room temperature for 20 minutes.
3. Rinse sections in 2 changes of washing buffer, 2 minutes each.
4. Serum Blocking: incubate sections with normal horse serum blocking solution for 30 minutes to block non-specific binding of immunoglobulin.
5. Primary Antibody: incubate sections with Mouse Anti-Rat CD54 (Pharmingen, Cat# 550302) diluted 1:50 in primary antibody dilution buffer for 1 hour at room temperature.
6. Rinse in washing buffer for 2x2min.
7. Peroxidase Blocking: incubate sections in peroxidase blocking solution for 10 minutes to block endogenous peroxidase activity.
8. Rinse in washing buffer for 3x2 min.
9. Secondary Antibody: incubate sections with biotinylated Horse Anti-Mouse IgG diluted in secondary antibody dilution buffer for 30 minutes at room temperature.
10. Rinse in washing buffer for 3x2min.
11. Detection: incubate sections with HRP-Streptavidin diluted in HRP-streptavidin dilution buffer for 30 minutes at room temperature.
12. Rinse in washing buffer for 3x2min.
13. Chromogen/Substrate: incubate sections in DAB peroxidase substrate solution for 5-10 minutes.
14. Rinse in distilled water briefly.
15. Counterstain with Gill's hematoxylin solution or Mayer's hematoxylin solution if desired.
16. Rinse in running tap water for 5 minutes.
17. Dehydrate through 95% ethanol for 2 minutes, 100% ethanol for 2x3min.
18. Clear in xylene for 2x3min.
19. Coverslip with permanent mounting medium.
Results:
Staining pattern: membrane/cytoplasmic of endothelial cells (Search Images)
