Are H4 cells good for imaging microtubules?

Hi everyone! I am trying to transduce NIH3T3 cells with lenti virus at a titer of 10^-8 with 24 well plate. I have tried multiple MOIs (5, 2, 1 , 0.5) which have all resulted in cell death. I originally attributed this death to titer concentration, and have transduced with 1:10 and 1:100 dilutions which still has lead to the same results (72 hour transduction) I will also mention that I have used polybrene solution at a 8 microgram/mL concentration to aid with efficiency for each trial. The control LV also looks wiped out but the non-transduction control has healthy cells. The next phase I would like to try reducing the incubation time to 24 hours, removing the polybrene and virus media, and then allowing the cells to grow for an additonal 2 days with fresh DMEM. Is this a good place to start/ has anyone else worked with NIH3T3 cells to transduce with lenti virus? is the polybrene toxic to NIH cells? Any suggestions are much appreciated !!

I want to stain the microtubules with a rabbit antibody anti-alpha tubulin through immunofluorescence (IF), to visualise the mitotic spindle of mouse and human cells. In our lab we had great experience with the mouse version of Abcam but to establish co-stainnings with other proteins, we would like to have an anti-rabbit or anti-goat version working. We tried out the one from Sigma (reference: SAB4500087) but the staining was not very good.

Could you help me to find out another option?

Thanks in advance,

Anchel

Hi everyone,

I am struggling with visualisation of possible deposition of cholesterol crystals in tissues of gastrointestinal tract.

I am wondering what method tu use, as there is a staining with filipin but from what I undestood - this will stain ALL OF FREE cholesterol (so also this deposited in cells), and I am not sure if that will make crystals vivible.

Second option - is using simply polarized light microscope, and here I would like to ask if anyone has experience or protocol he could share?

best!

They are visible on the surface showing that they are binding to Kinesin. I tried using different concentrations of ATP (even prepared fresh ATP) but no success. Could anybody please suggest something?

I'm interested in fixing and imaging cells after staining their microtubules (live, of course), so I'm looking for a microtubule stain for which fluorescence signal is not lost after fixation.

I am trying to compare the invasiveness among to cell types I am working with via a transwell invasion assay. I am used to counting manually under the microscope, something which I think can be unreliable sometimes and takes too much time. Can you recommend other reliable methods of quantification that can be done easily? Thank you.

I need some advice on the following experiment. We have fixed cells on coverslips that we need to examine with three specific antibodies. One is rabbit, two are mouse.

I can stain the rabbit with anti-rabbit alexa 488, and I can stain the first mouse antibody with anti-mouse alexa 594. After washing, I also want to have a third mouse antibody, but this antibody (to gamma-tubulin) will be conjugated directly to Alexa 647.

Can I do this? Obviously I am worried about the anti-mouse 594 going to the tubulin antibody, or that some of the tubulin might bind to the mouse 594 at that location. I haven't done 3 (4 with dapi) colour microscopy before....

I wanted to know how I could count the number of nerves in cryosections of the cornea after staining with Beta III tubulin. What software is available for automatic or manual counting?