10X Phosphate buffered saline (PBS) (Invitrogen 70011044)
PBST 1 x PBS plus 0.05 % Tween 20 (Sigma-Aldrich P9416)
PBST+TSA+DS: dissolve TSA blocking reagent (Perkin Elmer FP1020) at 1 %(w/v) in PBST, at 50ºC in water bath. Sterilise through 0.45 μm filter, and aliquot into 15 ml Falcon tubes, stock at -20ºC. Add normal donkey serum (Sigma-Aldrich D9663) and 1 M sodium azide to give 1 % and 10 mM, respectively.
Dulbeccos Modified Eagle Medium (DMEM): dissolve a bottle of powder DMEM (Sigma-Aldrich D5030-10xL), 4.5 g/L glucose, and 3.7 g/L NaHCO3 into milli-Q water, sterilise through 0.22 μm filter. Add 584 mg/L L-glutamine (Invitrogen 25030081) and 110 mg/L sodium pyruvate (Invitrogen 11360070). Store at 4ºC.
Mouse tonic saline: 0.6 % NaCl in milli-Q water, autoclave.
Rat Serum: Prepare according to ref . Alternatively, obtain from Equitech Bio Inc. (Equitech Bio,Inc. SRT-0010x10HI).
Foetal Bovine Serum (Invitrogen, routine cell culture grade)
Fluorescent dextran, M. W. 70,000 MW, aldehyde-fixable (Invitrogen D-1822 or D-1818): Dissolve into mouse-tonic saline at 25 mg/ml, dispense into small aliquots (25 μL in a microtube), keep at -20ºC, protect from light.
Anti-mouse IgG antibody (Jackson ImmunoResearch 715-095-151, 715-165-151, etc): Reconstitute at 2X concentration as specified by the manufacturer. Gently mix for at least 10 min, and check all the powder goes into solution, then add the same volume of glycerol, swirl well. Store at -20ºC, protect from light.
4% formaldehyde in PBS, freshly prepared from paraformaldehyde (PFA/PBS): weigh c.a. 1 g of paraformaldehyde (PFA)(Sigma-Aldrich, 158127) into a 50 ml Falcon tube, add c.a. 15 ml milli-Q (from a small vessel dedicated to this use only, in order to avoid reverse contamination of PFA powder and vapour into milli-Q stock for other critical experiments). Heat to c.a. 70ºC in a water bath with occasional swirling. If the powder does not go into solution, add a drop of 1 M NaOH, and swirl gently. Add 1/10 volume of 10 x PBS and adjust volume with milli-Q (again, from the small vessel). Place the tube on ice to cool, and use within 24 hours for primary fixation (i. e., before immunostaining or fresh tissues), or within a week for post-staining fixation (i. e., after 2nd antibodies incubation).
Gellan gum (Sigma-Aldrich P8169)
Vectashield mounting medium (Vector Lab H-1000)