FoxP3 Antibody - BSA Free

FoxP3 Antibody:
Protocol for Foxp3 staining.

- Cut 5 mm sections in a cryostat and fix them in cold acetone for 10 minutes
- Dry the slides completely at room temperature (30 minutes)
- Permeabilize tissues by immersing them in 0.1% saponin, 3 times (5 minutes each time)
-Block unspecific binding to the slides using 5% normal donkey serum, 5% normal rat serum and Fc block (1:500). Everything has to be dissolved in 0.1% saponin
-Incubate slides in a wet chamber at RT for 30 minutes
-Dip off the blocking solution from slides and add streptavidin (0.1 mg/ml) for 15 minutes at RT
-Wash one time with 0.1% saponin
-Add a similar volume of biotin (0.1 mg/ml) and incubate 15 min at RT
- Wash again with 0.1% saponin
- Add rabbit anti-Foxp3 (1:150) and biotin-rat anti-mouse CD4 (1:100) and incubate ON at RT
- Next day: Wash slides with PBS and add SA-594 or SA-488 (dissolved in PBS) to detect CD4 T cells (Dilution 1:500 from molecular probes). Incubate 1 h at RT
-Wash one time with 0.1% saponin and add biotin-donkey anti rabbit (Fab, Jackson Immunoresearch). Secondary antibody is diluted in PBS 1:500 and incubated by 3 hrs.
- Wash with saponin again and add SA-488 or SA-594 (1:500 in PBS). Incubate 1 h at RT
- Wash with PBS 3 times (10 minutes each wash)
- Mount slides with prolong gold antifade (Molecular probes)

Control: Incubated only with CD4 and no Foxp3
Tissue: thymus from an Influenza-infected mouse or thymus from healthy mice (C57BL/6)

Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes (keep slides in the sodium citrate buffer at all times).

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in PBS for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1% BSA in PBS) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul HRP polymer conjugated secondary antibody. Incubate 30 minutes at room temperature.
7. Wash sections three times in wash buffer for 5 minutes each.
8. Add 100-400 ul DAB substrate to each section and monitor staining closely.
9. As soon as the sections develop, immerse slides in deionized water.
10. Counterstain sections in hematoxylin.
11. Wash sections in deionized water two times for 5 minutes each.
12. Dehydrate sections.
13. Mount coverslips.

Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturer's instructions.