Thanks Richard!



-----Original Message-----

From: DICTY [mailto:[log in to unmask]] On Behalf Of Richard Gomer

Sent: 21 February 2020 15:37

To: [log in to unmask]

Subject: Re: [DICTY] fixing streams for microscopy



Hi-

This should work-

Coat a Millipore EZ slide with 20 mg/ ml bovine collagen or human vitronectin in 0.9% NaCl, 37˚ C 1 hour, then wash several times with PBS then 20 mM phosphate buffer or PBM (a buffer containing Mg and 10 micromolar Ca++;  note streams don’t form in the presence of higher levels of Ca++ according to Bill Loomis and from my observations)



Dave Knecht nice streams for microscopy:

Take an 8 well slide and add 300 microliters of PBM with 2 x 105 total cells per well (this is 300 microliters of 6.7 x 105 cells /ml) let settle, wait 45 minutes or so, remove 150 microliters from the top get wonderful streams a few hours later



 

Or to watch things without having to shock the cells by centrifugation just before development, on this surface, the evening/ afternoon before fixing, put dicty on at ~ 1/10 confluency in HL5, let grow overnight

Next day gently remove liquid, add PBM to well, gently remove, gently add PBM (500 microliters to well of a 4-well slide, 200 microliters to the well of an 8 well slide) – have PBM isothermal with slide 



If you have the slide in a little egg poacher pan, you can keep everything isothermal and minimize convection currents

 

To fix, without removing liquid from cells (this causes cells to skitter all over the place), gently add (1 part 37% formalin solution/9 parts PBM) to fill well 

after 20 minutes, gently remove and gently add more formaldehyde/PBM

after 10 minutes gently remove and add PBS/0.05% Tween-20

after 5 minutes change to PBS/ 0.02% NP-40 substitute



cheers



Richard Gomer



________________________________________

From: DICTY [[log in to unmask]] on behalf of Lenn, Tchern [[log in to unmask]]

Sent: Friday, February 21, 2020 8:56 AM

To: [log in to unmask]

Subject: [DICTY] fixing streams for microscopy



Hello,



Does anyone have any special techniques for fixing streams or the like for immuno-fluorescence?



Best

Tchern





Dr Tchern Lenn

Chubb Lab

MRC-LMCB

UCL