TITLE: Isolation of single somas from postmortem fresh frozen human brain and immunostaining for AT8 and MAP2
AUTHORS: Marcos Otero-Garcia, Inma Cobos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">- Protocol optimized to isolate single neuronal somas with cytoplasmic protein aggregates from postmortem fresh frozen brain</div><div class = "text-block">- Works for the isolation of  neuronal somas with neurofibrillary tangles from Alzheimer disease's brains</div><div class = "text-block">- Note that cell membranes are highly disrupted in tissues that were previously frozen. Thus, cytoplasmic transcripts are largely lost. This protocol is therefore useful for the isolation of specific cell populations using antibodies against cytoplasmic antigens, but not for the profiling of cytoplasmic mRNA.</div></div>

[STEPS]
?. Prepare Homogenization Buffer and cool it on ice.  ABC1AmountReagentFinal concentration22955 µlIM133 µlDTT 1mM1 µM430 µl50x Protease Inhibitor0.5 x515 µlRNaseIN 40 U/µl0.2 U/µl6No Triton!
ABC1AmountReagentFinal concentration22955 µlIM133 µlDTT 1mM1 µM430 µl50x Protease Inhibitor0.5 x515 µlRNaseIN 40 U/µl0.2 U/µl6No Triton!
?. Pre-cool the dounce tissue grinder on ice  (Potter-Elvehjem tissue grinder, 8 mL tubes, 0.1–0.15 mm clearance space between pestle and tubes).
?. Add  of Homogenization buffer to the dounce tissue grinder.
2.4 ml
?. Collect the brain chunk (~) and transfer to a Petri dish on ice.
200 mg
?. Cut out into small pieces using a chilled scalpel or blade.
?. Transfer all pieces into the dounce tissue grinder.
?. Slowly homogenize the tissue, on ice (8~15 strokes; slow). Check on hematocytometer while homogenizing and adjust the number of strokes.
Too few strokes – you will see clumps; too many strokes – you will get a higher proportion of naked nuclei.
?. Filter homogenate using a 100 µm Corning cell strainer to remove clumps.-->  Take sample for hemocytometer.
?. Transfer the homogenate into two precooled 1.5 ml Eppendorf tubes.
?. Centrifuge at  for  at .
4 °C
?. Slowly aspirate the supernatant from the side of the tube. Avoid disturbing the pellets, up to  of supernatant can be left.
50 µl
?. Gently resuspend each pellet in  (final volume) of cold Homogenization buffer and pool both tubes (final volume ).
225 µl
450 µl
?. Add an equal volume of cold 42% v/v iodixanol.
Critical! Be very precise with volumes.   suspension +   50% iodixanol. Final iodixanol concentration is 21%.
?. Gently pipette mix.
?. Add an equal volume of 25% iodixanol () into a 2 ml Eppendorf tube.
900 µl
?. Slowly layer off the 21% iodixanol suspension mix over the 25% iodixanol, without mixing them.
?. Centrifuge at  for  at .
4 °C
?. Prepare buffer for Immunostaining.  ABC1AmountReagentFinal concentration29400 µlRNase-free PBS (pH7.4)3500 µlRNase-free 10 % BSA0.5 %450 µl1 M MgCl25 mM510 µlDNAse I (2000 U/ml)2 U/ml
ABC1AmountReagentFinal concentration29400 µlRNase-free PBS (pH7.4)3500 µlRNase-free 10 % BSA0.5 %450 µl1 M MgCl25 mM510 µlDNAse I (2000 U/ml)2 U/ml
?. After centrifugation, remove and discard the top myelin-rich debris layer (you can use a 1 ml pipette with the tip cut, or cotton swabs).
?. Remove and discard the aqueous supernatant, without disrupting the somas pellet. Avoid contaminating with the top layer.
?. Use a small amount of buffer for Antibody Incubation to resuspend the pellet and transfer the solution to a new tube.
?. Gently resuspend in  of buffer for Antibody Incubation.
200 µl
?. Incubate for , at  or iced, for blocking nonspecific staining.
4 °C
?. Take sample for hemocytometer.
?. Take a sample for unstained control. Take sample for 2AB-only (nonspecific binding control).
?. Add primary antibodies (Ms-a-AT8 1:150 and Rb-a-MAP2 1:40) and incubate on a shaker in cold room () for .
4 °C
Use Eppendorfs coated with BSA for collection (to coat the Eppendorf tubes, fill them with 10% BSA solution in PBS for  , rinse with PBS, and dry at   overnight).
?. [Washing]
Add  of buffer for Immunostaining and invert several times.
500 µl
?. [Washing]
Centrifuge at  for  at .
4 °C
?. [Washing]
Carefully transfer all the supernatant (don’t leave any supernatant! It’s ok to drag some pellet) to a new Eppendorf tube and resuspend the pellet in  of buffer for Antibody Incubation.
400 µl
?. [Washing]
Centrifuge the supernatant at  for  at  to recover non-pelleted somas.
4 °C
?. [Washing]
Resuspend them in  of buffer for Antibody Incubation and pool for a final volume of 600 µl.
200 µl
?. Take samples for single color staining.
?. Add secondary antibodies (G-a-Ms Alexa350, 1:500 and G-a-Rb Alexa647, 1:500) and Sytox Green 1:400 (1:100 stock, final concentration 1:40.000)
?. Check the quality of the sample on hemocytometer.
For 200 mg of tissue, we should have ~ 1-2 Million somas in ~0.6 mL.
?. Transfer to 7 ml culture tubes.
?. Place on ice and bring them to the FACS facility for sorting.
FACS sorting controls:Non-stained2AB onlySingle stainings
?. Collect in BSA-coated Eppendorfs containing collection medium (volume of collection medium should be ~1/5 of the expected final volume after collection).
?. Add BSA after collection for 1 % BSA final concentration (tested for 10x Chromium v2 and v3).
Different Single Cell assays tolerate different BSA concentrations, but lowering it may increase nuclei aggregation.
?. Add .
[RNase-free PBS (pH7.4)]
?. Add .
[RNase-free 10 % BSA]
?. Add .
[1 M MgCl2]
?. Add .
[DNAse I (2000 U/ml)]
?. Add .
[IM1]
?. Add .
[DTT 1mM]
?. Add .
[50x Protease Inhibitor]
?. Add .
[RNaseIN 40 U/µl]
?. Prepare buffer for Antibody incubation (1.5 ml per sample) by adding   of RNaseIN 40 U/µl to  of buffer for Immunostaining (final concentration 0.2U/µl)
5 µl
1 ml