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

[Brian W. Jones]

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