Material:
1) Bouin's Fixative Reagent
2) Peroxiclase Block Reagent
3) 20X Phosphate Buffered Saline
4) Remaining reagents are found in the HISTOSTAIN SP Kit
Methods:
1) Prepare the sections/slices as described by the protocol and store at -20 C until usage
2) Remove the sections from the freezer and air dry for 20 minutes
3) Add Bouin's fixative reagent on the surface of the sections for 20 minutes
4) Wash quickly in 1X PBS and then soak in 1X PBS for 2 x 5 minutes
5) Soak in Methanol: Peroxide solution for 30 minutes
6) Soak in 1X PBS for 3 x 10 minutes
7) Add 2 drips of serum blocking solution to the surface of the section and leave for 1 hour
8) Dilute the primary antibody to the appropriate concentration and add 2 drips to the surface of the section and leave for 2 hours. Additional drops may be required, it is important not to let the section dry. Blot dry the section.
9) Soak in 1X PBS for 3 x 5 minutes
10) Apply 2 drips of the secondary antibody to the surface of the section and leave 10 minutes. Blot dry the section.
11) Soak in 1X PBS for 3 x 5 minutes.
12) Add 2 drips of enzyme conjugate to cover the section and leave for 10 minutes.
13) Soak in 1X PBS for 3 x 5 minutes.
14) Add 1 drip of the AEC substrate to 1 mL of water and apply to the survace of the section for 20 minutes. From this step forward, the section needs to be protected from light. Rinse well with dd H20.
15) Counterstain with hematoxylin for 30 sec - 1 minute. Wash with tap water.
16) Place slides into 1X PBS until they turn blue.
17) Mount the slides
| Reagent | Final Conc. | Volume | Mass |
| NaCl | 160 g | ||
| KCl | 4 g | ||
| NA2HPO4 | 28.8 g | ||
| KH2PO4 | 4.8 | ||
| H2O | 800 mL |
pH to 7.4. Adjust volume to 1L. Autoclave.