IHC-Paraffin and IHC-Frozen Protocol
I. Preparation of slides:
1) Deparaffinization: soak tissue slides with xylene twice, each time for 15 minutes
2) Rehydration: incubate slides in the following graded series of ethanol: 100%I, 100%II, 95%, 90%, 80%, 70%. 5 minutes for each solution. Then incubate in the water for 5 minutes.
Note: The step 1 is only for paraffin embedded slide. For frozen slide, soak the slide in water for 5 minutes, and go to step 2.
II. Protocol for Immunostaining:
1) Immerse the slides in 0.3% H2O2 (in distilled water) for 30 minutes at room temperature
2) Rinse the slides with water followed by 1x PBS (pH7.4) once, circle the tissue section with a Pap Pen (or Para-Pen, ImmEdge Pen)
3) Incubate the slide with 1% Normal serum/PBS [Mix 3.5 ml 1x PBS, pH 7.4 with 1 drops (about 35 ul/drop) of normal serum in a tube] for 30 minutes at room temperature
4) Drop off the normal serum from the slides
5) Incubate the sections with the PBS diluted first antibody (optimize the antibody titer before starting IHC) in a humid chamber for 1 hour to overnight at room temperature.
6) Rinse the slides with 1xPBS for 3 times, each time for 5 minutes
7) Incubate the slides with PBS diluted Biotin-labeled secondary antibody for 30 minutes at room temperature. [Mix 1.4 ml 1xPBS pH 7.4 and 1 drop (about 35 ul/drop) of biotinylated anti-mouse IgG in a tube]
8) Rinse the slides with 1xPBS for 3 times, each time for 5 minutes
9) Preparation of detection solution: Mix 1.33 ml 1xPBS, 1 drops (about 35 ul/drop) of Solution A, and 1 drops (about 35 ul/drop) of Solution B in a tube. Incubate the mixture at room temperature for 30 minutes before use.
10) Add the detection solution prepared at step 9) to the tissue section. Incubate room temperature for 30 minutes.
11) Rinse the slides with 1xPBS for 3 times, each time for 5 minutes.
12) Make fresh Development solution: Mix 1.6 ml DAB buffer, and 1 drop (about 35 ul/drop) of Liquid DAB in a tube.
13) Add the Development solution to cover the tissues, and develop for 5-30 minutes
14) Stop the reaction by soak the tissue in water.
15) Counter stain the slides if necessary (Use Harris' Hematoxylin to stain the nuclear, and use eosin Y to stain cytosol)
16) Dehydrate by soaked in the graded series of alcohol: 70%-80%-90%-95%-100%I-100%II, 3 minutes for each solution; then incubated in Xylene twice, 10 minutes each.
17) Mounting the slides (Fisher's permount Cat. # SP15-100 or similar mounting material can be used)
18) Add 2 drops (about 35 ul/drop) of detoxification solution into the used development solution. Incubate for 1 hour or overnight. Then it can be discarded.