Contents |
MA, USA).
Burlingame, CA, USA).
(DAB; Sigma).
Novabiochem, San Diego, CA, USA).
PA, USA).
(Baxter Healthcare, McGraw Park, IL, USA).
From Watson et. al. (44a). 1.59 g NaH2PO4 .H2O (mono) 5.47 g Na2HPO4 (dibasic) 9.0 g NaCl 300 g Sucrose 10 g Polyvinyl pyrrolidone (PVP-40) (Bio-Rad) 300 mL Ethylene glycol Bring to 1 L with distilled water and store in refrigerator. Procedure 1. To fix the sections on the filters, 4% formaldehyde/PBS is placed beneath and on top of the slice on the Millicell filter for 1.5 to 2 hours. 2. The slice cultures are thoroughly but gently rinsed with PBS. If rinsed too hard, it might dislodge and damage the tissue. 3. Use a scalpel blade or scissors to cut out the filters carrying the slices. 4. Place the filters holding the slices in Netwells into the 6-well plate carriers containing PBS (or into cryoprotectant if you are not going to stain tissue right away). Note: It is very important that the slices on the filters are always fully immersed throughout this and all following procedures. 5. Rinse the slices extremely well in PBS before going into blocking solution. 6. Place the slices into blocking solution (10% NGS, 0.3% Triton X-100, 0.01% Na azide) for 1 to 2 hours. 7. Place into primary antibody (diluted in 1% egg albumin, 0.01% Na azide) and incubate overnight at 4°C. 8. Rinse well in PBS (3 × 10 min) using Netwells and carriers. 9. Apply secondary antibody—rabbit or mouse IgG biotinylated (at 1:500 dilution) in PBS at room temperature for 1.5 to 2 hours. Do not add sodium azide to this mixture, as it will interfere with the peroxidase reaction. 10. Rinse well in PBS (3 × 10 min). 11. Place slices (in Netwells) in ABC solution in the ABC kit (1:600 dilution) in the 6-well plates for 1 to 1.5 hours at room temperature. 12. Rinse tissues well with 2× PBS and rinse with 2× TBS (if you are using NiSO4 in DAB reaction mixture). 13. Place sections (in Netwells) into the DAB reaction mixture. To make DAB reaction mixture: Imidazole 136 mg NH4Cl 40 mg Glucose 400 mg PBS 85 mL or TBS 85 mL (when using with NiSO4) To the above, add 10 mL of filtered DAB (100 mg DAB/20 mL in 1× TBS, pH 7.4). Then add 500 μL of glucose oxidase (100 U/mL) to above and mix well. Important: Allow mixture (with or without 70–80 mg NiSO4/100 mL) to sit at least 10 to 15 minutes before use. 14. Gently rock sections (in Netwells) in DAB reaction mixture (time of reaction should be determined empirically). 15. Stop reaction by rinsing thoroughly in PBS (2 × 10 min) or TBS and finally in fresh PBS. 16. Samples can be stored in PBS no longer than 2 days. 17. If you are doing double label IHC, do the NiSO4 step first (this results in a black reaction product). Then rinse well in PBS (3 x 10 min) and block again for 30 minutes in NGS. 18. Place sections into the second primary antibody and incubate overnight at 4°C. 19. Repeat the above steps 8 through 15. The final staining reaction for the second antibody is in the same DAB reaction mixture, but without NiSO4 (reaction product is brown in color).
1. Set Netwell in water before removing tissue from filter. 2. Place a drop of water on the coated slide. 3. Place the filter on top of a petri dish in a drop of water. 4. Gently ease the tissue off with the camel hair brush. 5. Let the tissue stick to the brush and place it in the drop of water on the slide. 6. Check the slide under the scope after you have removed all of the tissue and gently unfold and straighten out the tissue. 7. Remove excess fluid with filter paper. 8. Allow to air-dry undisturbed overnight. 9. Place slides in water for 15 to 20 minutes to remove salt. 10. Wash in 100% EtOH (2 × for 10–15 min). 11. For slides only, clear in xylene (3 × for 10–15 min). 12. Add permount and place coverslip on slides.
Note: In some cases, the tissue may be
too fragile and might have to stay on the filter
in order to be processed. In these cases,
place sections still on filters and in Netwells
into Americlear Clearing Solvent to clear
tissue (xylene will cause filters to curl up).
Remove section still on filter and place filter
on slide, add permount, and place coverslip
over filter containing the section.
Visualization of the immunostained cells
will be useful but less than optimal as compared
to the mounted section that can be
completely removed from the filter.
<comments />