ImmunoHistoChemistry of KRS using mouse tissue

 

 

1.     Warm the slides at 60degree for at least 30 min

2.     Deparaffin the slides at Xylene 4 times for each 10 min.

3.     Rehydrate the slides 100% ehanol, 100% ethanol, 90%, 80%, 70% for each 2 min

4.     Rinse the slides in DW 2 times for each 5 min

5.     Immerse the slides into citrate buffer (0.01M pH 6.0)(make 500ml)

-Use slide rack and yellow tip box

-Pour the buffer 350ml into tip box and soak the slide rack

-run the micro wave for 5 min, and 1 min cool  at RT

-Repeat 3 times above step (total 4 times)

-If the buffer is vaporing out, supplement buffer with residual 150ml

Stock :0.1M citrate-A, 0.1M sodium citrate(trisodium citrate dihydrate)-B

Working Sol-pH 6.0: A- 9ml + B-41ml +450ml DW=> final 500ml

6.     Immerse the slides in cold PBS for 10 min

7.     Quenching the endogenous peroxidase using 3% hydrogen peroxide sol for 10 min (Sigma H 6520)

8.     Wash the slides PBS for 5 min, 3 times.

9.     Block the slides with 2% FBS in PBS for 1hr in 37 degree

10.  Incubate the slides with primary antibody (1/20 diluted serum in blocking sol) for 1.5 hrs at 37 degree

11.  wash slides for 5 min X4 times with PBS

12.  Incubate the slides with biotin- conjugated secondary(rabbit) Ab for 1 hr at RT

13.  repeat step 11

14.  Incubate the slides with streptavidin-HRP for 20 min at 37 degree

15.  repeat step 11

16.  substrate reaction for 2-10 min at RT

17.  put the slides into slide rack within DW box & exchange the DW with fresh and stand it for 5 min.

18.  Hematoxilyn staining for 5 sec in 1/2 diluted hematoxylin

19.  DW staining to remove the residual hematoxylin.

20.  mounting & watching