[Histonet] extensive troubleshooting of IF on fixed-then-frozen mouse brain

[Jonathan Cremer]

You're not making your formaldehyde solution properly, that might be the first of your issues.
Paraformaldehyde is the polymer of formaldehyde, and has little to no fixative properties. It doesn't dissolve in water either. To make a proper fixative from paraformaldehyde, you have to hydrolyse it in a basic solution.

To make 4% formaldehyde in PBS, resuspend 40 g of paraformaldehyde in about 700 ml of water and add one or two drops of 2-3 M NaOH. Leave it stirring for 5-10 minutes, add one drop of NaOH if the solution hasn't cleared up entirely. (You can warm up the liquid to speed to process, but don't go above 60 °C or the formaldehyde will start to decompose.) Don't overdo it with the NaOH, because you have to bring down the pH again with HCl, and the more you add, the more the salinity of the final solution will go up.
When you have a clear solution, add either 100 ml of 10x PBS, or add the dry powders for 1 l of PBS. Stir to dissolve/mix, then bring the pH to 7,4 (or whichever desired) with 1 M HCl. Finally, add water to a total volume of 1 l.

To my view, the PBS is there mostly to buffer the formic acid from formaldehyde decomposition. You could make up in water or saline only, but keep an eye on the pH.

Also, when diluting concentrated formaldehyde (formalin and formal are just old commercial names for 37-41% of formaldehyde in water, with 10% methanol), use PBS for immediate use (the small polymers are then hydrolized rapidly) or dilute in water and leave 48 hours before use (slow depolymerization).
---
Jonathan Cremer
Laboratory Technician
TARGID - KU Leuven

Gasthuisberg CDG; Labo Experimentele Immunologie; Herestraat 49 bus 811; 3000 Leuven; Belgium

________________________________________
Van: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] namens Brian W. Jones [bwjones <@t> uw.edu]
Verzonden: dinsdag 5 februari 2013 0:38
To: histonet <@t> lists.utsouthwestern.edu
Onderwerp: [Histonet] extensive troubleshooting of IF on fixed-then-frozen      mouse brain

Hi All,

I've used Histonet many times to answer my IF/IHC questions, but this
time I'm stumped. We've been using a transcardial perfusion
fixation/immunofluoresence protocol for years and it has always worked.
It has worked so reliably that we would often make last-minute
changes---e.g., post-fix overnight instead of 4 hours---if something
else came up and it would still turn out fine.

Well, obviously things aren't working now or I wouldn't be pleading for
help!

THE PROBLEMS:
Mouse brain tissue has large holes (Swiss cheese look), lacks fine
structures such as axons or dendrites (looks mushy), some of the brain
regions easily separate away from others (e.g., the hippocampus
separates from the thalamus), and random areas of the section are not in
the same focal plane as the surrounding tissue. (I tried to post images
as instructed on the web site, but I can't figure that out. Please
advise as I think the images would show better than I can describe.)

THE PROTOCOL:
Here is a detailed description of the protocol and variations I have
tried while troubleshooting. Note that I have not tried every
combination of every variation, and certainly I am careful to only
change one thing at a time so I can make meaningful comparisons.

1) Euthanize 3 - 5-month old mouse with Beuthansia D. Wait until no
longer responsive to front paw pinch and restrain animal on dissecting
board. Make incision to expose thoracic cavity and lift ribcage out of
the way. Heart is still beating strongly at this point.
2) Grasp heart with blunt forceps and a) make small incision in left
ventricle and insert 20-g cannula, or b) insert 20-g or 25-g needle into
left ventricle.
3) Using a peristaltic pump, perfuse PBS and immediately cut right
atrium for outflow. Have tried PBS at either 4 C or 35 C. Have made up
PBS fresh and checked pH. Have replaced tubing and used a different pump.
4) Set flow rate to a) 1.5 mL/min, b) 3 mL/min, c) 6 mL/min, or d) 10
mL/min. (Do not have a way to measure pressure.)
5) Perfuse PBS: a) 3 mL only, b) until liver blanches (never really
blanches at low flow rates), or c) 15 mL.
6) PBS is either a) PBS alone, or b) PBS with heparin.
7) Perfuse with 50 mL of fixative: 4% PFA in PBS or PLP (Nakane)
fixative. Note: I have tried PFA from Sigma (two different lots) or EMS
(granules or 16%). Fixative is perfused at same rate and temp as PBS.
8) For 8% PFA stock: PFA is usually dissolved in water but have tried
directly in PBS. Dissolved by bringing solution to 55 C then adding PFA
while stirring. A few drops of 1-4 M NaOH are added until solution
clears (a few undissolved particles remain). Solution is filtered
through Whatman paper or 0.45 um membrane. Always made fresh the day of
the perfusion.
9) Animal's lower extremities begin to move shortly after introduction
of fixative. Rarely observe perfusate exiting the nose/mouth.
10) Post-fix: after perfusion, brain is dissected from skull using
scissors and forceps and placed in 4 C fixative for 4-7 hours or overnight.
11) Brain is cryoprotected in sucrose-PBS: a) 10% sucrose until brain
sinks, then 20%, then 30%; or b) 30% sucrose until brain sinks; c) three
changes of 25% sucrose over three days total, or d) some other variation
that ultimately ends in a brain sunk in at least 25% sucrose.
12) Brain is placed in 4 C Tissue-Tek OCT and frozen a) on powdered dry
ice, or b) liquid nitrogen and then on dry ice while freezing other brains.
13) Brain is stored at -80 C or taken directly to cryostat. Have used
two different cryostats.
14) Brains are allowed to equilibrate to cryostat temp, usually -17 C.
15) 20 um sections with new knife are a) placed thawed directly onto
slide or b) placed in PBS and floated onto slide. Note: everything
"feels" right on the cryostat.
16) Slides: a) VWR Superfrost, b) Fisher Superfrost Plus, or c)
Superfrost Plus Gold.
17) Sections on slides are a) left to air dry for 30+ minutes, b) stored
-80 C, or c) placed immediately in TBS-T.
18) Antibody procedures are done in 1% BSA in TBS-T; washes are with
TBS-T. (Have tried using another lab's TBS-T as well.)
19) Slides are mounted with Prolong Gold + DAPI --- and by this time
I've gone through several bottles of it, so it's never old!

As mentioned above, in the past it seemed that any variation to the
protocol would still work. Now, it seems that I can't make it work no
matter what I change. I was convinced that it was a fixative problem,
but as noted I've tried multiple sources/preparations of PFA. As my
colleague put it: "You've tested all the scientific and all the
superstitious factors."

Thank you for any help you can provide!

--
Brian Jones, Ph.D


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