Alexa 488 conjugated antibodies

Tissue preparation for immunofluorescence was performed as previously described [2 (link)]. Anesthetized animals were transcardially perfused with 4% (vol/vol) paraformaldehyde in phosphate-buffered saline pH 7.4, then tissues were cryopreserved in 30% (wt/vol) sucrose, and embedded in TissueTek OCT (Sakura). Following cryosectioning of spinal cord into 30 μm-thick sections, free floating sections were selected from the same well per animal for each experiment and immunostained with the indicated antibodies. In brief, free floating sections were washed in PBS three times before incubation in blocking solution containing PBS with 1.5% (wt/vol) BSA and 0.5% Tween-20 for 1 h at room temperature. Sections were incubated in primary antibodies (detailed in Supplementary Materials) in PBS, 0.3% (wt/vol) Triton-X 100 overnight at room temperature, and then washed three times in PBS before incubation with secondary antibodies in PBS, 0.3% (wt/vol) Triton-X 100 for 1 hr at room temperature. After incubation with DAPI (1 μg/ml) and a final wash with PBS, sections were mounted onto glass slides and allowed to dry overnight before coverslipping with ProLong Gold anti-fade mounting media (Invitrogen). For detection of primary antibodies, donkey anti-rabbit, anti-mouse, or anti-goat Cy3, Cy5, or Alexa-488 conjugated antibodies (Jackson ImmunoResearch) were used at a 1:500 dilution.

Animals (n = 5/6 animals per group) were perfused through the heart with ice-cold saline, followed by a solution of 4 % paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, via the ascending aorta. The spinal cords were then removed, and cryoprotected by immersion in 20 % sucrose followed by 30 % sucrose in phosphate buffer. The spinal cords were sectioned transversely at 20 μm on a cryostat (CM 1850; Leica, Wetzlar, Germany) and collected on gelatin-coated slides.
For immunohistochemistry, sections were washed three times with PBS with 0.3 % Triton X-100, incubated with 5 % normal goat serum (NGS; Sigma) in PBS for 30 minutes, and then incubated with the primary antibodies monoclonal mouse anti-NeuN (1:200, #MAB 377; Chemicon, Temecula, CA, USA) and polyclonal rabbit anti-Iba1 (1:400, #019-19741; Wako, Richmond, VA, USA) overnight at 4 °C. After the primary incubations, the appropriate secondary Cy3®-conjugated or Alexa® 488-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA and Invitrogen Inc., Carlsbad, CA, respectively) were used. In some cases, the sections were incubated with TO-PRO®-3 (1:1000; Invitrogen) for nuclei staining. Sections were mounted with VectaShield® (Vector, Burlingame, CA, USA) and analyzed using confocal microscopes (LSM 510 META or LSM 510 META NLO; Zeiss GmbH, Oberkochen, Germany).