Mouse anti β 3 tubulin

Primary cortical neuronal cultures derived from C57Bl/6 and Tau KO mice (Tau KO primary neurons only used for LDH) were prepared and maintained as previously described [74 (link), 84 (link), 92 (link)]. The primary antibodies used in this study for immunocytochemistry are as follows: rabbit anti-I11 [55 (link)], mouse anti-β-III-Tubulin (Abcam #78078), rabbit anti-total p53 (Abcam #246550), rabbit anti-total tau (Abcam #64193), mouse anti-phosphorylated p53 (Ser15) (Cell Signaling #9284), and rabbit anti-phosphorylated histone H2AX (Ser139) (Cell Signaling #2577). After three washes with PBS, cells were probed with mouse and rabbit-specific fluorescent-labeled secondary antibodies (1:200, Alexa Fluor 568, Life Technologies). The single-frame images and Z-stacks for orthogonal view were collected using a Keyence Confocal Microscope. To build the Z-stack, 17 stacks/0.3–0.4-μm optimal thickness were captured. Each treatment condition was randomly imaged in five different regions of interest and performed in duplicate. Imaging processed with ImageJ Software (NIH).

The selected slides were placed in an oven at 60°C to remove excess paraffin, dehydrated in a series of graded alcohols, and placed in a Coplin jar with 10 mM citrate buffer at pH 6, inside a pressure cooker for 20 min to perform antigen recovery. Then, the tissues were permeabilized with PBS-T (0.01 M PBS and 0.1% Triton) for 30 min. Non-specific sites were blocked with 5% horse serum in PBS-T in a humid chamber for 30 min, and the tissues were incubated with goat anti-GFAP (1:500, Abcam), rabbit anti-cleave caspase-3 (1:300, Cell Signaling), and mouse anti–βIII-tubulin (1:500, Abcam) primary antibodies in a humid chamber at 4°C overnight. Afterward, the tissues were washed with PBS-T and incubated with Alexa Fluor^® 488 donkey anti-rabbit IgG (1:500, Molecular Probes), Alexa Fluor^® 594 donkey anti-goat IgG (1:500, Molecular Probes), and Alexa Fluor^® 405 donkey anti-mouse IgG (1:500, Molecular Probes) secondary antibodies for 2 h at room temperature in the dark. Then slides were placed in a solution of 0.1% Black Sudan B (Sigma-Aldrich) in 70% ethanol for 15 min, the excess Black Sudan B was removed using distilled water and PBS-T. The tissues were covered with VECTASHIELD^® and a coverslip and were observed under a confocal Nikon TI eclipse microscope.

For embryonic tissues, freshly dissected intestines were fixed 1 hour at RT with 4% PFA in PBS. Alternatively, whole embryos were fixed overnight at 4°C and the intestines dissected afterwards. For adult tissues, whole intestines were fixed in 4% PFA overnight at 4°C, cut longitudinally along the mesentery and washed in PBS. The outermost muscle layers were then stripped from the mucosa/submucosa. Fixed tissues were dehydrated in methanol. After rehydration, the intestines were incubated 2 hours at RT in blocking solution (10% fetal bovine serum, 0.1% Triton-X100 in PBS). Tissues were incubated with primary antibodies overnight at 4°C. The antibodies used were: mouse anti-βIII-Tubulin (1:200; Abcam ab78078), rabbit anti-S100β (1:500; Dako Z0311), goat anti-Sox10 (1:100, Santa Cruz Biotech. sc-17342) and rabbit anti-Ki67 (1:1000; Abcam ab15580). Secondary antibodies Alexa Fluor 594- or Alexa Fluor 647-conjugated anti-goat, -mouse or -rabbit (1:500, Jackson Immunoresearch) were incubated for 2 hours at RT and counterstained with DAPI. All antibodies were diluted with blocking solution. For the TUNEL assay, tissues were permeabilized 20 min at 37°C in 0.3% Triton-X100, 0.1% sodium citrate in PBS 1x, then stained in a 1:9 mix of enzyme solution:label solution from the in situ cell death detection kit, TMR red (Roche Applied Science 12156792910) 1h at 37°C.