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Collagen  Type  1a1  -­‐  Immunohistochemistry  Protocol    

 

MaxPoly-­‐Two  Polymer  HRP  Mouse  Detection  Kit  (w/DAB)  Cat#  PT02-­‐D      
For  Formalin  Fixed  Paraffin  Embedded  Rabbit/Mouse  Tissue  

Cut  sections  at  3-­‐5  microns      

 

DAY  1  
 
 1.      Bake  slides  at  60°C  for  60  minutes  prior  to  starting  IHC.      2.    Deparaffinize  slides,  thrice  in  xylenes  for  5  min  each,  and  rehydrate  through  graded  alcohols.              (100%  EtOH  twice,  95%  EtOH  twice  and  70%  EtOH  once  for  5  min  each)      3.      Wash  twice  in  deionized  water  for  5  minutes  each.      4.        Pap  around  sections.      5.      Quench  in  Dako  Duel  Endogenous  Enzyme  Block  (Dako  S2003)  for  20  minutes.      6.      Rinse  in  PBS  -­‐  3  times  for  5  minutes  each.      7.      Enzymatic  Digest  –  Dako  Proteinase  K  Digestion  (Dako  S3004)  –  Mix  one  drop                                              (40uL)  in  2mL  0.05mol/L  Tris-­‐HCL  pH  7.5  to  7.7.  Incubate  at  ROOM                        TEMPERATURE  for  10  minutes.      8.      Rinse  in  PBS  -­‐  3  times  for  5  minutes  each.      9.    Use  1:2000  dilution  of  Anti-­‐Collagen  1  antibody  [COL-­‐1]  (ab90395)  in  either  DAKO                  Diluent  (DAKO  S0809)  or  MP  Normal  Antibody  diluent  (980641)  and  incubate                overnight  at  ROOM  TEMPERATURE.      

 

         

6. 7.3 times at 5 minutes each. Incubate with Rabbit Antibody Amplifier from MaxPoly-Two Polymer HRP Rabbit Detection Kit (w/DAB) (PT02-D) for 15 minutes. Counter stain slides in Hematoxylin (Zymed Cat #93-3943) for 1 minute.DAY 2 1. Alcock.S. Detect color with DAB from MaxPoly-Two Polymer HRP Rabbit Detection Kit (w/DAB) (PT02-D). Standardized on Mouse tissue on 12/19/2011 by Ashish Thomas. Takes 2-5 minutes checking under microscope.3 times at 5 minutes each. Rinse slides in PBS . 10. 8. Mix 50ul DAB chromogen (Reagent 3A) to 1mL DAB substrate buffer (Reagent 3B). Rinse slides in PBS . Rinse slides in PBS . Clear in 3 changes of Xylene – mount with Cytoseal. Incubate slides with Polymer HRP from MaxPoly-Two Polymer HRP Rabbit Detection Kit (w/DAB) (PT02-D) for 15 minutes. Dehydrate through 3 changes of 95% ethanol and 2 changes of 100% ethanol for 1 minute each. Rinse in TAP water until clear. 4. .3 times at 5 minutes each. 3. M. Reagent Lot numbers: Proteinase K: DAKO S3004 MaxPoly-Two Polymer HRP Rabbit Detection Kit (w/DAB): PT02-D DAKO Diluent: DAKO S0809 or MP Biomedicals: Normal Antibody Diluent: 980641 Anti-Collagen 1 antibody [COL-1]: Abcam – ab90395 Standardized on Rabbit tissue on 12/8/2011 by Nanette C. 9. 11. 2. 5. Rinse in Deoinized water to stop reaction for 3 minutes.