[Histonet] CD 31 for rabbit

[Galina Deyneko]

Bernice,
   CD 31 mouse anti-human Dako #M 0823 works great in my hands on FFPE and frozen rabbit's aorta. On frozen I do not use pre-treatment. 
  My protocol:
            IHC protocol for rabbit’s tissues using CD31 and Polymer –HRP. 
  1.De-wax and hydrate the slides.
  2 Place slides in Tris Buffer 
  4. Preatretment with Proteinase K for 15 min. at RT
  W.Volume :  10 000µl)
  º (Promega # V3021)
     Concentration: 10mg/1ml (100mg is diluted in 10 ml of 1mM Tris (Ambion #9856,pH8.0)
      Final concentration: 50 µg/1ml 
      Preparation: 10000 µg of concentration : 50 µg of final concentration = 200 diluent factor. Dilution of Proteinase K 1:200.
  3.000  µl working volume : 200 =  15 µl of concentrated Proteinase K + 2.985  µL of 1  mM Tris Buffer.
   Preparation of 1mM of  Tris Buffer: 1 µl of  1M Tris Buffer + 999 µl of D water
                                                              3 µl of 1M Tris + 2.997 µl of D water 
  Optional: Proteinase K (Dako, cat.# S3004)
                   40 µl ( 1 drop)  of concentrated proteinase K mix with 4 ml of  0,05 M Tris, pH 8 (manufarcture instruction)
  2 drops of concentrated proteinase K with 10 ml of 0,05 M Tris
     Preparation of 0,05 mol/L Tris:
  1M Tris x X µl = 0,05 M Tris x w.volume:
  1M Tris x X µl = 0,05 M Tris x 10 000 µL
  X = 0,05M x 10 000 µl : 1M = 500 µl of 1M Tris + 9500 µl of D water
  5. Rinse with Tris Buffer 
  6. Rinse with Tris Buffer.
  7. Block peroxidase activity with Peroxidazed 1 for 5 min. at RTº. (Biocare #PX968)
  8. Rinse with Tris Buffer 
  9. Protein block with Background Sniper for 20 min at RTº ( Biocare #BS966).
  10. Rinse with Tris Buffer 
  10.Incubation with primary AB overnight at 4 C ( Monoclonal Mouse anti-Human CD 31, Endothelial cell, Dako Cytomation #M0823)
        Dilution 1:100
        Diluent: Da Vincy Green, Biocare #PD900
  8.500 µl working volume :100 =85µl of AB 
  85µl of primary AB +8415 µl of diluent.
  Day two 
  11. Rinse with Tris Buffer.
  12. Incubation with MACH 2 Poly HRP Conjugate  for 30 min. at RTº (Biocare, # MHRP520).
  13. Rinse twice with Tris Buffer 
  14.Developing with DAB for     min at RTº   (Biocare # DB801,kit)
      1000 µl of Substrate Buffer  + 50 µl (1 drop) of Chromogen
  15.Rinse with water.
  16.Developing with DABSparkle for 2 min at RTº (Biocare, # DS830)
  17. Counterstain with Gill’s Hematoxylin dilution 1:5 (Poly Scientific, cat# s211)for    min
  18. Dehydrate, coverslip.
   
  Galina Deyneko
  Novartis ,Cambridge MA 

 
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