Protocol
Immunohistochemistry
Chen lab@NCCU
1) Deparaffinate by xylene twice, each for 5min.
2) Gradient rehydrate: 100%, 95%, 70% and 50% ethanol, each for 2min.
3) Wash with running tap water for 2min.
4) Prepare AUS (3ml + 200 ml water) and heat it for 2min. Keep the slides in AUS for 5min. Heat with a microwave or pressure cooker for 5min.
5) Wash with running tap water for 2min.
6) Wash with PBS twice for 3min each.
7) Treat with 3% H2O2 for 30 min at RT.
8) Wash with running tap water for 2min.
9) Wash with PBS twice for 3min each.
10) Block with 1% to 10% normal serum for 30min at RT.
*Mouse Ab: Horse NS *Goat Ab: Rabbit NS
*Rabbit Ab: Goat NS * Sheep Ab: Horse NS
11) Incubate with primary antibody, dilute properly in normal serum, at RT overnight.
12) Wash with running tap water for 2min.
13) Wash with PBS thrice for 3min each.
14) Incubate with biotinylated secondary antibody (1:200 NS) for 30min at RT.
15) Wash with running tap water for 2min.
16) Wash with PBS thrice for 3min each.
17) Incubate with ABC (5ml PBS + 2 drops A and B, or ready-to-use ABC) for 30min at RT.
18) Wash with running tap water for 2min.
19) Wash with PBS thrice for 3min each.
20) Incubate with DAB (1 DAB tablet + 1 Urea/H2O2 tablet with 5ml water) under microscope.
21) Wash with running tap water for 2min.
22) Counter stain with Meyer’s hematoxylin for 1 to 4 min.
*If using Harris hematoxylin, stain for 4sec, wash with running tap water.
*Differentiate with acidic alcohol, wash with running tap water for 30min.
23) Wash with running tap water for 10min.
24) Dehydrate with 50%, 70%, 95% and 100% ethanol, each for 2min.
25) Clear with xylene, twice for 2min each.
26) Mount with mounting media.
Hematoxylin and Eosin staining
1) Deparaffinized with xylene, 2 times for 5 min each.
2) Rehydrated via gradient ethanol,100%->95%->70%->50% for 2 min each.
3) Washed in running tap water for 5 min..
4) Incubate in Harris Hematoxylin for 1 min.
5) Washed in distilled water, 2 times for 5 min each.
6) Dipped in acid alcohol, 3 times for 1 sec each.
7) Washed in running tap water for 15 min.
8) Dipped in Eosin solution, 4 times for 1 sec each.
9) Rehydrated via gradient ethanol 50%->70%->95%->100% for 2min each.
10) Cleared with xylene, 2 times.
11) Mounted.
Agarose/Paraffin Triple Embedding Method
Tissue for processing
Alcian Blue/ PAS Staining
1) Deparaffinized by xylene 1 and 2 – 5min each.
2) Dehydrate by gradient ethanol, concentration 100% -> 95%-> 70%->50% - 5min each.
3) Wash with running tap water for 5 min.
4) Treat with 3% acetic acid for 2 min.
5) Treat with Alcian blue solution ( 1gm of Alcian Blue, 86x, in 3ml acetic acid and 97 ml of distill water) for 30min.
6) Wash with running tap water for 2 min.
7) Wash with distilled water, 2 changes for 2 min each.
8) Incubate in peroxide acid at room temperature for 10 min.
9) Wash with running tap water for 5 min.
10) Wash with distilled water, 2 changes for 2 min each.
11) Incubate in Schiffs reagent at room temperature for 10 min.
12) Wash with 0.5% potassium metabisulfite (1gm in 200 ml of distilled water) , 3 changes for 2 min each.
13) Wash with distilled water for 5 min.
14) Dehydrate with gradient ethanol, 50%->70%->95%->100%, for 5 min each.
15) Cleared by xylene 1 and 2 for 5 min each.
16) Mounted.