Niemann-Pick type C1 Like-1 Antibody - BSA Free

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

Western Blot Protocol

1. Perform SDS-PAGE (3-8%) on samples to be analyzed, loading 50ug of total protein per lane.
2. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer apparatus.
3. 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% non-fat dry milk in TBS for 1 hour at room temprature.
6. Dilute the rabbit anti-NPC1L1 primary antibody (NB 400-127) in 5% BSA and incubate overnight at 4 degreesCelcius.
7. Wash the membrane in water for 5 minutes and apply the diluted rabbit-IgG HRP-conjugated secondary antibody inblocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
8. Wash the blot in TBS containing 0.05-0.1% Tween-20 for 10-20 minutes.
9. Wash the blot in type I water for an additional 10-20 minutes (this step can be repeated as required to reducebackground).
10. Apply the detection reagent of choice in accordance with the manufacturers instructions (Amersham's ECL is the standard reagent used at Novus Biologicals).

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