>>>Cell Surface Immunofluorescence Staining Protocol
1.Harvest desired cells and adjust cell suspension to a concentration of 1x10⁶ cells/ml in PBS/BSA. Whole blood samples could also be used.
2.Aliquot 100 ul of cell suspension/whole blood into required number of test tubes.
3.Add appropriate volume of prediluted fluorochrome conjugated antibody. Mix well and incubate at room temperature for 20 minutes.
4.If using separated cells follow step 5.
5.If using whole blood samples, add 2 ml of freshly prepared Genemay RBC-lysing solution (Cat. No. GMH801) and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 300g for 5 minutes. Discard supernatant.
6.Wash with 2 ml of PBS/BSA, centrifuge at 300g for 5 minutes. Discard supernatant.
7.Resuspend cells in 0.5 ml of PBS/BSA or with 0.5 ml of 1% paraformaldehyde in PBS/BSA if required.
8.Acquire data on a flow cytometer

>>>Intracellular Cytokine Immunofluorescence Staining Protocol
1.Harvest desired cells and adjust cell suspension to a concentration of 1x10⁶ cells/ml in PBS/BSA. Whole blood samples could also be used.
2.Aliquot 100 ul of cell suspension/whole blood into required number of test tubes.
3.If performing cell surface antigen staining at the same time, add appropriate volume of prediluted fluorochrome conjugated antibody first. Mix well and incubate at room temperature for 20 minutes.
4.If using separated cells follow step 5.
5.If using whole blood samples, add 2 ml of freshly prepared Genemay RBC-lysing solution (Cat. No. GMH801) and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 300g for 5 minutes. Discard supernatant.
6.Wash with 2 ml of PBS/BSA, centrifuge at 300g for 5 minutes. Discard supernatant.
7.Add 0.5 ml/tube Fixation Buffer (Cat.No.GMH808), mix well and incubate in the dark for 20 minutes at room temperature.
8.Add 2 ml/tube Permeabilization solution (Cat.No.GMH809) and centrifuge at 300g for 5 minutes. Discard supernatant.
9.Repeat step 8 once.
10.Resuspend cells in residual Permeabilization solution and add appropriate volume of fluorochrome conjugated anti-cytokine antibodies or an appropriate isotype control for 20 minutes in the dark at room temperature.
11.Wash twice with 2 ml Permeabilization solution and centrifuge at 300g for 5 minutes. Discard supernatant.
12.Resuspend cells in 0.5 ml of PBS/BSA.
13.Acquire data on a flow cytometer.

>>>Western Blotting Protocol
1. Place desired cells in a microcentrifuge tube and centrifuge to collect cell pellet.
2. Lyse the cell pellet with 100 µl lysis buffer on ice for 30 minute.
3. Centrifuge at 14000 rpm for 10 min at 4^oC.
4. Transfer the supernatant to a new tube and discard the pellet. Remove 20 µl supernatant and mix with 20 µl 2Xsample buffer.
5. Boil for 5 min. Cool at RT for 5 min. Microcentrifuge for 5 min.
6. Load up 40 µl of sample to each well of a 1.5 mm thick gel.
7. Set gel running conditions and transfer the proteins to a nitrocellulose or PVDF membrane with variable power settings according to the manufacturer/'s instructions.
8. Remove the blotted membrane from the transfer apparatus and immediately place in blocking buffer consisting of 5% nonfat dry milk/TBS-T.
9. Incubate the blot for 1 hour at room temperature, or overnight at 4^oC with agitation.
10. Dilute the primary antibody to the recommended concentration/dilution in 5% nonfat dry milk/TBS-T. Place the membrane in the primary antibody solution and incubate 2 hours at RT, or overnight at 4⁰C with agitation.
11. Wash three times for 5 min each with Wash Buffer (TBS containing 0.1% Tween-20).
12. Incubate the membrane for 30 min at RT with HRP conjugated secondary antibody, diluted to 1:1000 in 5% nonfat dry milk/TBS-T.
13. Wash 4 times for 10 min each with TBS containing 0.1% Tween-20, and once for 2 min with PBS.
14. Incubate membrane (protein side up) with 10 ml ECL for 1-2 min. The final volume required is 0.125 ml/cm2.
15. Drain off excess detection reagent, wrap up the blots and gently smooth out any air bubbles.
16. Place the wrapped blots, Protein side up, in a X-ray film cassette and expose the x-ray film. Exposures can vary from 5 seconds to 60 min.

>>>IHC Staining of Frozen Tissue Sections
1. Embed fresh tissues carefully in OCT in plastic mold, taking care not to trap air bubbles surrounding the tissue. Freeze tissue by setting mold on top of liquid nitrogen until 70-80% of the block turns white. Then, put block on top of dry ice. Frozen blocks may be stored at -80^oC for long-term storage.
2. For cutting step, mount the frozen block on the cryostat holder. Never, at any point, let the tissue warm up to temperatures above minus 15^oC.
3. Allow frozen blocks to equilibrate in the cryostat chamber for about 5 minutes. Cut 6-10mm sections. The best sections are usually obtained when the block temperature is around minus 18 to minus 20^oC.
4. Let the sections dry for at least 30 minutes at room temperature. Note: Upon drying, tissues can be stored at 4^oC for a few days; for longer-term storage up to a few months, store at -80^oC.
5. Fix the sections by immersing in acetone jar for 1-2 minutes at room temperature, and let air-dry. Carefully draw boundaries of sections using a PAP Pen and let it dry for a few minutes.
6. Add primary antibodies (diluted in 0.05M Tris-Saline, pH 7.4, 2.5% serum) directly onto the sections. Add in big droplets to cover the entire tissue at once to prevent fracturing of the sections due to the surface tension of the solution. Incubate in a chamber for at least one hour at room temperature. Note: Once the sections are re-hydrated with Tris-saline, never let the tissue dry out again (this will ruin the tissue architecture).
7. Wash the sections gently in Tris-saline for 3-5 minutes and then in Tris-saline/2.5% serum for another 3-5 minutes.
a. If using biotinylated primary antibodies, skip the following 2 steps (8 and 9) and move to step 10.
b. If using purified primary antibodies, continue the protocol at step 8.
8. Without letting the sections dry out, add the secondary antibody diluted in Tris-saline/2.5% serum. Incubate as before for at least 45 minutes.
9. Wash as described in step 7.
10. Cover slides with about 100µl of diluted SA-AP or SA-HRP. Incubate for 30 minutes at room temperature.
11. Wash as described in step 7.
12. Stain slides.
a. For HRP, incubate with AEC substrate: 25µl stock AEC (4mg/ml in DMSO) per 1ml 0.17 M NaOAc, pH 5.2 plus 1µl H₂0₂. Incubate for 20-30 minutes.
b. For AP, incubate with AP substrate: Fast Violet (1mg/ml final) + Napthol AS-MX phosphate (0.2mg/ml final, from 10mg/ml stock in DMSO) in Tris-Saline pH 8.5 for 10-20 minutes until the desired positive staining is achieved.
13. Wash 2 times in Tris-saline.
14. Counter-stain with Mayer/'s hematoxylin for 30 seconds and wash with tap water for 2-5 minutes.
15. Mount coverslips with mounting media.
16. Read the slides.

>>>General Protocol for the Sandwich ELISA method
1. Before the assay, both antibody preparations should be purified and one must be labeled.
2. For most applications, a polyvinylchloride (PVC) microtiter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.
3. Bind the unlabeled antibody to the bottom of each well by adding approximately 50 µl of antibody solution to each well (20 µg/mL in PBS). PVC will bind approximately 100ng/well (300 ng/cm2). The amount of antibody used will depend on the individual assay, but if maximal binding is required, use at least 1µg /well. This is well above the capacity of the well, but the binding will occur more rapidly, and the binding solution can be saved and used again.
4. Incubate the plate overnight at 4^oC to allow complete binding.
5. Wash the wells twice with PBS. A 500 mL squirt bottle is convenient. The antibody solution washes can be removed by flicking the plate over a suitable container.
6. The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Fill the wells to the top with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 hrs. to overnight in a humid atmosphere at room temperature.
7. Wash wells twice with PBS.
8. Add 50 µl of the antigen solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS). Incubate for at least 2 hrs. at room temperature in a humid atmosphere.
9. Wash the plate four times with PBS.
10. Add the labeled second antibody. The amount to be added can be determined in preliminary experiments. For accurate quantitation, the second antibody should be used in excess. All dilutions should be done in the blocking buffer.
11. Incubate for 2 hrs. or more at room temperature in a humid atmosphere.
12. Wash with several changes of PBS.
13. Add substrate as indicated by manufacturer. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA plate reader. (For quantitative results, compare signal of unknown samples against those of a standard curve. Standards must be run with each assay to ensure accuracy.)