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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
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