Hi Mimo Sa

I use a concentration of 0.2% T-X100 for 15 mins in room temp. That works well for me to stain mitochondrial proteins.

Thanks

Samir

Hi Samir,

Thanks for your help. I usually do 10 min paraformaldehyde fixation at room temperature? Do you recommend to fix it longer to stain mitochondrial proteins?

Samir Ranjan Panda

Hi Mimo,

For macrophages and neuronal cells, I used a 10min PFA fixation while for other cells (cardiac, cancer) I fix them for 15mins. We can optimise it for our cell by visualising in microscope.

Thanks

Hi Samir Ranjan Panda ,

Thanks for your helpful answers. Could you please explain how we could determine by microscope whether the cells were sufficiently fixed or needed more fixation? Is there is any criteria? By the way, would you mind sharing your standard ICC staining procedure?

Thanks

Hi Mimo Sa

Because of the abrupt change in osmolarity of media and fixative solution, I have observed that some cells like macrophages (RAW 264.7) and microglia change their morphology and appear to be swollen when fixed for longer period. So while using fixative for the first time, I would recommend you to observe the cells in microscope post adding PFA, If until 15mins you don't find any morphological differences then you can proceed with staining.

Please find the protocol attached. Do let me know if there are any further queries.

Protocol

1. cells were plated at a density of 2×106 cells/well on poly-D-lysine coated coverslips in a six-well plate.

2. Following treatment, cells were washed with PBS, fixed with 4% PFA for 15 mins, permeabilized using 0.2% Triton X-100 for 10 mins, and blocked with 5% normal goat serum (NGS) for 1 hr.

3. Post blocking, the cells were incubated with primary antibodies overnight at 4°C.

4. The cells were washed with PBST for 10mins, Next, the cells were incubated with secondary antibody for 1hr in dark.

5. Following secondary, cells were washed in PBST for 10mins and then mounted using mounting media with DAPI in a clean slide.

6. The images were captured in confocal microscope.

Thanks

Samir

Hi Samir Ranjan Panda ,

Thanks for your prompt and helpful answers. Regarding the protocol:

what is the recipe of antibody dilluent you use?

Second, is there a difference between fixation using cold (4 degree) versus room temperature PFA?

Thanks

Hi Mimo Sa

I dilute the primary antibody (dilution recommended according to antibody company) in PBS and 0.1% Tween, and secondary I use secondary antibody, Alexa-Flour at a dilution of 1:1000 in PBS and 0.1% Tween.

I do not use cold fixative solution for cells, i use it in room temp as it is milder and does not affect the morphology of the cells, i used cold one for tissue fixation.

Thanks

Samir

Hi Samir Ranjan Panda ,

Thank you. One last question, is the 5% NGS blocking buffer prepared in 1XPBS or it is better to add tween or tritonx-100 and at what concentration?

Thanks

Hi Mimo Sa

5% NGS in 1X PBS and add 0.1% T-X100. (So for example if we want to prepare 10ml Blocking solution, Take 0.5ml NGS, 10ul T-X100 and dissolve in 9.5ml of 1X PBS). Blocking Time-1hr at room temp.

Thanks

Samir