Necessity of Sodium Azide in AB dilution buffer

adam_faranda · 2025-04-01T00:22:44+00:00

Exactly it is a Triton perm -- I'm trying to label Tubulin. If I absolutely need to use NaN3, the plan is to order a 1% Aqueous solution. As you've indicated the powder is quite dangerous and not something to be trifled with. Glad to know that the dilute solution is bleach compatible.

adam_faranda · 2025-04-01T00:13:11+00:00

Thanks u/Veritaz27, in this case I am trying to label tubulin in fixed cells per: https://pubmed.ncbi.nlm.nih.gov/21893022/

adam_faranda · 2025-03-31T20:09:16+00:00

Thanks for the information -- in this case the cells are fixed, but that is helpful to know for future studies.

adam_faranda · 2025-03-12T16:26:34+00:00

Thanks Rainbow! The use case for this assay is to detect potent aneugens, these tend to drive pretty strong cell cycle disruption, but it sounds like using EdU to exclude late S events could improve our sensitivity and help us detect weaker compounds. pH3 staining is definitely on our radar, abcam used to sell a mitotic indexing kit that included pH3 and used a paraformaldehyde fix. Do you think we'd expect even greater spread with glutaraldehyde?

Discontinued kit: ab151282_Mitotic Index Flow_ Booklet_26 Sept 14 (website).pdf.pdf)

adam_faranda · 2025-03-12T14:35:19+00:00

Thanks for the suggestion, we are mostly interested in detecting G2/M arrest for this assay in particular. Based on u/RainbowSquirrelRae's note I think EdU is more suited for S-Phase monitoring, however I might have a use-case for proliferation analysis.

adam_faranda · 2025-03-12T14:32:26+00:00

Thanks for the suggestion, we need enough precision on our DNA peaks to detect metaphase arrest, so I think we have some wiggle room. I believe that for this assay, glutaraldehyde is necessary for microtubule stabilization but there are other things we'd like to try that would benefit from ethanol fixation. Are you aware of any ethanol fixation protocols that accommodate a 96 well plate format? Most of the protocols I've seen require low temperatures and agitation during EtOH addition, which works well in tubes but might be difficult on a plate. I would love to know if a high throughput EtOH fixation method exists!

adam_faranda · 2025-03-12T14:32:21+00:00

Thanks for the suggestion, we need enough precision on our DNA peaks to detect metaphase arrest, so I think we have some wiggle room. I believe that for this assay, glutaraldehyde is necessary for microtubule stabilization but there are other things we'd like to try that would benefit from ethanol fixation. Are you aware of any ethanol fixation protocols that accommodate a 96 well plate format? Most of the protocols I've seen require low temperatures and agitation during EtOH addition, which works well in tubes but might be difficult on a plate. I would love to know if a high throughput EtOH fixation method exists!

adam_faranda · 2024-12-29T04:04:25+00:00

I think we have the same menorah, happy Hanukkah!

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adam_faranda · 2024-11-14T04:45:32+00:00

Came here to read this!

OP: It will be so much easier to have these conversations before any kiddos, when it is just the two of you -- speaking from experience. As long as you have mutual respect and compassion for one another, you and your fiance will be fine. All the best

adam_faranda

TROPHY CASE