Anti calb1

For IHC, tissue was fixed with 4% PFA and cryoprotected with 15%/30% sucrose gradient as previously described prior to storage at −80° C and cut at 50pm using a freezing microtome [21 (link)]. Floating IHC was performed as previously described [21 (link)] with whole rabbit antisera anti-Calb1 (Cell Signaling Technology, Inc, Beverly MA; 1:250), or anti-GFAP (DAKO / Agilent Technologies, Santa Clara, CA; 1:1000) followed by goat anti-rabbit antibody (1:200) used as the secondary antibody and DAB. Cresyl-violet (CV) staining was performed to assess injury at P11, P18 and P40 and for volumetric analysis as described ahead. Antibodies. Calb1 (CS13176; RRID:AB_2687400): Rabbit monoclonal antibody raised against a recombinant protein specific to the N-terminus of whole Calb1 protein of human origin detecting product at 28 kDa (2μg/ml). GFAP (DAKO Z0334; RRID:AB_10013382): Rabbit polyclonal antibody raised against GFAP isolated from cow spinal cord with no reported cross reactivity (1μg/ml).

For IHC, pups were killed with an overdose of isoflurane as described above, and exsanguinated with cold 0.1 M PBS (pH7.4) via intra-cardiac perfusion. Brains were perfusion fixed with 4% PFA in 0.1M PBS for 30 min at 4ml/min. Tissues were cryoprotected with graded immersion in 15% and then 30% sucrose in PBS until the tissue sank, frozen and stored in −80° C until cut at 50μm on a freezing microtome (Northington et al., 1996 (link)). Floating IHC was performed as previously described (Northington et al., 1996 (link)) with whole rabbit antisera anti-PV (ABcam plc, Cambridge, MA; 1:250), anti-Calb1 (Cell Signaling Technology, Inc, Beverly MA; 1:250), anti-GFAP (DAKO / Agilent Technologies, Santa Clara, CA; 1:1000), or anti-GAD65/67 (ABcam PLC; 1:500) followed by goat anti-rabbit antibody (1:200) used as the secondary antibody and DAB as the chromagen (Northington et al., 1996 (link)). CV counterstaining was performed in those sections previously immunostained for PV to better assess the nuclear morphology of pyramidal cells surrounding PV+INs.