LC3A Antibody - BSA Free

Immunocytochemistry Protocol

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

1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three times for 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to wash solution at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.

*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.

Protocol: Inhibition of Autophagy and LC3 Antibody (NBP1-19167) Western Blot

Materials

Chloroquine diphosphate (CQ) (10 mM) in dH2O
1X PBS
Sample buffer, 2X Laemmli buffer: 4% SDS, 5% 2-mercaptoethanol (BME), 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris HCl, pH 6.8
RIPA buffer: 150 mM NaCl, 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 20 mM Tris-HCl, pH 7.5
1X Running Buffer: 25 mM Tris-base, 192 mM glycine, 0.1% SDS. Adjust to pH 8.3
1X Transfer buffer (wet): 25 mM Tris-base, 192 mM glycine, 20% methanol, Adjust to pH 8.3
TBS
TBST, TBS and 0.1% Tween
Blocking solution: TBST, 5% non-fat dry milk
rabbit anti-LC3 primary antibody (NBP1-19167) in blocking buffer (~2 ug/mL)

Methods

Tip: For more information on Western Blotting, see our Western Blot handbook.

1. Grow cells (e.g. HeLa or Neuro2A) in vitro to semi-confluency (70-75%).

2. Add CQ to culture dishes to a final concentration of 50 uM and incubate overnight (16 hours). Remember to include an untreated sample as a negative control.
Note: Validated autophagy inducers should be included as positive controls.

3. Rinse cells with ice-cold 1X PBS and lyse cells with sample buffer.
Note: LC3-I and LC3-II are sensitive to degradation, although LC3-I is more labile. These proteins are sensitive to freeze-thaw cycles and SDS sample buffers. Fresh samples should be analyzed quickly to prevent protein degradation.

4. Sonicate and incubate cells for 5 minutes at 95oC.
Tip: Cells are lysed directly in sample buffer or may be lysed in RIPA buffer.

5. Load samples of Chloroquine-treated and -untreated cell lysates 40 ug/lane on a 4-20% polyacrylamide gradient gel (SDS-PAGE).
Tip: For detection of LC3 it is particularly important to monitor the progress of the gel as this protein is relatively small (~14kDa).

Tip: Alternatively, for non-gradient gels, use a 20% polyacrylamide gel.

6. Transfer proteins to a 0.2 um PVDF membrane for 30 minutes at 100V.

7. After transfer, rinse the membrane with dH2O and stain with Ponceau S for 1-2 minutes to confirm efficiency of protein transfer.

8. Rinse the membrane in dH2O to remove excess stain and mark the loaded lanes and molecular weight markers using a pencil.

9. Block the membrane using blocking buffer solution (5% non-fat dry milk in TBST) for 1 hour at room temperature.

10.Rinse the membrane with TBST for 5 minutes.

11.Dilute the rabbit anti-LC3 primary antibody (NBP1-19167) (~2 ug/mL) in blocking buffer and incubate the membrane for 1 hour at room temperature.

12.Rinse the membrane with dH2O.

13.Rinse the membrane with TBST, 3 times for 10 minutes each.

14.Incubate the membrane with diluted secondary antibody, according with product's specifications, (e.g. anti-rabbit-IgG HRP-conjugated) in blocking buffer for 1 hour at room temperature.
Note: Tween-20 may 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.

15.Rinse the membrane with TBST, 3 times for 10 minutes each.

16.Apply the detection reagent of choice (e.g. BioFX Super Plus ECL) in accordance with the manufacturer's instructions.

17.Image the blot.
Tip: LC3-I and it's lipidated form LC3-II have different electrophoretic mobility properties, with the lipidated form moving faster in an SDS-PAGE gel, albeit its larger molecular weight. LC3-II runs at 14-16 kDa while LC3-I runs at 16-18kDa.

Note: This assay measures the difference in the LC3-II signal in the presence and absence of inhibitors (e.g., lysosomotropic agents). When autophagic flux is present or induced in a system an increase in the LC3-II signal should be observed with the inhibitor.