Pyruvate Carboxylase Antibody - BSA Free

Immunocytochemistry Protocol

Culture cells to appropriate density in 35mm culture dishes or 6-well plates.

1. Pull off culture medium with and add 10% formalin to the dish. Fix at room temperature for 30 minutes..
2. Take off the formalin and add ice cold methanol (kept in well sealed bottle in -20C). Incubate for 5-10 minutes.
3. Take off methanol and add PBS (You can add 0.1% Tween-20 to PBS used here and all subsequent steps), be sure to not let the specimen dry out. Wash 3 times 10 minutes before proceeding to blocking step.
4. To block nonspecific antibody binding incubate in 10% normal goat serum for a minimum of 1 hr at room temp. Cells can also block overnight at 4C for this step.
5. Add primary antibody at appropriate dilution and incubate at room temp for 2 hrs or overnight at room temp.
6. Remove primary antibody and replace with PBS. Wash 3 x 10 min in PBS.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hr at room temperature
8. Remove antibody and replace with PBS, wash 1 x 10 min in PBS. Add Hoechst 33258 to PBS at 1:25,0000 and incubate for 10 min. Wash a third time with PBS for 10 min (total of 3X10min PBS washes).
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide and parafilmed. Cells can also be coverslipped using Fluoromount. If storing coverslip be sure to seal the edges with clear nail polish.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.

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.

Staining:
1. Wash sections in dH2O three times for 5 minutes each.
2. Wash section in wash buffer (1X PBS/0.1% Tween-20 (1X PBST)) for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1X PBST, 5% goat serum) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul primary antibody diluted in 1X PBST, 5% goat serum to each section. Incubate overnight at 4C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated secondary antibody, diluted in 1X PBST, 5% goat serum. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
8. Add 100-400 ul Striptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in dH2O.
12. Counterstain sections in hematoxylin.
13. Wash sections in dH2O two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.

Western Blot Protocol
1. Perform SDS-PAGE (4-12% MOPS) on samples to be analyzed, loading 40 ug of total protein per lane.
2. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer apparatus.
3. Rinse membrane with dH2O and then stain the blot using Ponceau S for 1-2 minutes to access the transfer of proteins onto the nitrocellulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations and locations of molecular weight markers using a pencil.
4. Rinse the blot in TBS for approximately 5 minutes.
5. Block the membrane using 5% NFDM + 1% BSA in TBS + Tween, 1 hour at RT.
6. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.
7. Dilute the rabbit anti-Pyruvate Carboxylase primary antibody (NBP1-49536) in blocking buffer and incubate 1 hour at room temperature.
8. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.
9. Apply the diluted rabbit-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer [TBS + 0.1% Tween] 3 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 manufacturers instructions (Pierce ECL).

**Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%, provided it does not interfere with antibody-antigen binding.