Hello,
I would like to do immunostainings on differentiated 3T3-L1 cells (adipocytes). We tried most of the "classic" protocols, fixation with either 4% PFA or cold methanol/acetone, and a lot of commercially available antibodies. If most of these antibodies work very well with other cell lines, there is absolutely no staining in differentiated 3T3-L1 cells. We suspected an insufficient permeabilization of the cells but surprisingly, one custom antibody targeting a protein from the lipid droplet membrane works perfectly...
If you thus have suggestions or protocols adapted for differentiated 3T3-L1 cells, it would be greatly appreciated.
Hello Stephane,
For 3T3-L1 adipocytes immunostaining we usually follow the protocol from Abcam with minor modifications.
Briefly:
- Fixation using 4% Formaldehyde in PBS for 15 min, RT.
- Permeabilization with 0.25% Triton X-100 in PBS for cytoplasmic or nuclear antigens, for 10 min. We have also used 0.5% Saponin in PBS for detecting inner leaflet antigens.
- 1 hour blocking with 1% BSA, 22.52 mg/mL Glycine (important to get rid of excess formaldehye) in PBS.
- Primary antibody 1 hr 37 °C.
- Secondary antibody 1 hr RT.
Maybe this is more like a classic protocol, but it worked well in our experiments.
Hope it helps.
http://www.abcam.com/protocols/immunocytochemistry-immunofluorescence-protocol
Hi Stephane,
If you want to 4% PFA solution for fixation, fresh made 4% PFA solution is an important tip.
For permeibilzation (oil red O stain), 4 %PFA, 30 min at RT is enough.
For non permeibilzation (GLUT4 translocation assay by using myc-GLUT4-GFP), 4%PFA, less than 10 min, RT.
Hope it helps.
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