9:30a – 10:00p
Goals
- Need to finish review
- Need to make slides for symposium at Penn
- send revised
- Troubleshooting sudden failure in staining
Troubleshooting staining
Background of problem:
Stained new cells on Saturday, went to image these on Monday — no staining. Suspected extended hybe time could have been problematic, or something wrong with new multi-color probes. One idea I had about the extended hybe time was that the rubber cement seal is not very good and there’s a fair amount of condensation onto the slide, which may have leached out the probe concentration. I tried staining another three coverslips of cells using a probes that had previously worked and using a dry box. I still observed condensation and I still observed no staining. Now I thought I might have screwed up the cell prep, so I repeated the cell prep with a fresh batch of cells on Wed and stained these with E3 probes for which I very recently got beautiful staining. Yet again, no staining. To really see if this was hybe buffer / staining conditions or cell-prep, I took an old coverslip (previously stained for C8, a very small weak staining region) and stained again with the large / strong C1 probe. I used this same probe mix and hybe buffer to stain some of the cells prepped Wed, one with and one without RNase treatment. The C1 on the old cells stains great, the new prepped cells both no staining.
The cell prep is reasonably straightforward and I had been using all the same buffers and stuff for a while, so I’ve no explanation of why this should suddenly fail. I did notice a little growth in my PBS glycerol, which I promptly replaced. Yesterdays cells were briefly exposed to this before I noticed the tiny cloudy clusters in the glycerol. I have a hard time believing though that this brief exposure to some extra bacteria or mold eating the glycerol would completely and utterly destroy the ability to hybridize FISH probes to the genome…
All New Buffers
- made completely new PBS
- made new 1/2x PBST and 5% formaldehyde
- made new 0.5% Triton-X in PBS
- have new 20% glycerol in PBS (using old stock of 10x PBS though…)
- rinsed PBS squirt bottle. Maybe we should not use it for this round just to be sure.
- made new 0.1 M HCl from 37% stock
Area cleaning
- bleached and washed bench
- bleached and washed pipettes
- new pipette tips
- set up new clean-zone at desk with bench guard.
New Cell Culture
- started new cell culture from frozen stock.
- Probably enough cells here to plate 1 well and try staining? They may be a little pissed just coming out of LN2 today, but it’s a comparison.
New cell staining controls
- Resuspend new cells and plate in 4 wells
- Resuspend old cells and plate in 8 wells
- allow cells 1 hour to attach
- follow my protocol
- stain new cells with C1 probe (no RNase)
- stain old cells with C1 probe (no RNase)
- stain new cells (with RNase 32 min at 37 treatment) with E4 probe. This sample kept in separate dish for this step.
More control ideas:
- RNase treat C7 and then stain it with C1. (see if RNase treatment is a problem / contaminate). It is a new batch of RNase dilution (same original enzyme lot).