Cell Culture
Use standard culture protocols for maintenance of cells. For adherent cells, seed on sterile coverslips for 24-48 hours. For non-adherent cells, cells can be fixed in suspension and attached to poly-l-lysine coated coverslips before antibody staining.
For this protocol we used the adherent cell line PNT2 grown in RPMI media supplemented with 10 % fetal bovine serum. Cells were seeded at 1 × 105 cells / mL in 6 well plates and cultured for 48 hours.
Fixation
We recommend fixing with paraformaldehyde. Cold methanol fixation can be used, however this may reduce ER staining intensity.
- Fix cells in 4% paraformaldehyde in PBS pH 7.4 for 10 minutes at room temperature.
- Wash cells three times with PBS for five minutes each at room temperature.
Permeabilisation
- Incubate the samples for 15-25 minutes with 0.5% w/v saponin in PBS at room temperature.
- Wash cells three times with PBS for five minutes each at room temperature.
Blocking
- Incubate cells with 1% BSA, in PBST (PBS+ 0.1% Tween 20) for 30 min at room temperature to block non-specific binding of the antibodies
Antibody staining
- Prepare primary antibody to required concentration in 1% BSA in PBST. For this example we use rabbit anti-Cytochrome-C (Abcam ab90529 0.6m/mL) at a concentration of 1 μg/ml (for localisation to mitochondria).
- Incubate cells in the diluted primary antibody for one hour at room temperature or overnight at 4°C.
- Remove primary antibody and wash the cells three times in PBS for five minutes each.
- Prepare secondary antibody to required concentration in 1% BSA in PBST. For this example we used goat anti-rabbit Alexa Fluor 647 at a 1/1000 dilution.*
- Incubate cells with the secondary antibody for one hour at room temperature in the dark.
- Aspirate the secondary antibody and wash cells three times with PBS for five minutes each.
- CAUTION for best results select a secondary antibody with a fluorophore which is compatible with ReZolve-ER (ex. UV or 400 nm, em. 570 nm). We recommend a far-red fluorophore such as Alexa Flour 647. Whichever fluorophore you select ensure that it is not excited at UV or 400 nm excitation.
Endoplasmic Reticulum Staining
- Prepare 10 mM stock solutions of ReZolve-ER by dissolving the powder in DMSO as instructed on the vial.
- Prepare a staining solution by diluting the stock in PBS to 20 μM (1/500 dilution).
- Incubate cells with staining solution for 30 min at room temperature, in the dark.
- Remove coverslips from the staining solution and mount in aqueous mounting media. For best results ensure some of the staining solution is transferred into the mounting media.*
- CAUTION do not wash coverslips after ReZolve-ER staining. Washing will significantly reduce the fluorescence obtained from the staining.
Mounting and Imaging
- Once mounted adhere coverslips to the slide using quick-dry nail polish.
- For best results, imaging by confocal microscopy is recommended.
- For excitation of ReZolve-ER a UV, 385 nm or 400 nm light source can be utilized. For detection, a detector compatible with FITC is most appropriate, or if available a tuneable detector set to a detection range of 525 nm - 600 nm.
- For antibody imaging use appropriate settings for the selected secondary antibody. For this example we have used a 647 nm excitation and a detector compatible with far-red fluorophores.
- To avoid signal bleed through, sequential imaging is recommended.