Rabbit anti tuj1

ROCK1/2 and mTORC1 (Raptor) /C2 (Rictor) shRNAs were obtained from Millipore-Sigma. Western blotting analyses were performed to check the knockdown efficiency. Immunofluorescence staining was performed as follows: 5 × 10⁴ modified human fibroblasts were planted on Matrigel-coated glass coverslips the day before induction. Cells were fixed for 20 min at room temperature in 4% paraformaldehyde in PBS, permeabilized for 30 min in PBS containing 0.2% Triton X-100 and 10% normal goat serum (NGS) and incubated overnight at 4 °C in PBS containing 10% NGS and primary antibodies. Cells were washed three times with PBS and incubated for 2 h at room temperature with anti-rabbit or anti-mouse secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-594 (1:500, Invitrogen). Images were acquired on immunofluorescence microscope or Zeiss LSM 510 META confocal microscope at 40× magnification and 1.3 numerical aperture oil-immersion objective.
The following antibodies were used for the immunofluorescence studies: rabbit anti-MAP2 (Millipore-Sigma, 1:200), mouse anti-Tuj1 (R&D Systems, 1:100), rabbit anti-synapsin 1 (Cell Signaling, 1:200), mouse anti-Tuj1 (1:1000, Covance) and rabbit anti-Tuj1 (1:2000, Covance).

Cells were fixed in 4% paraformaldehyde for 10 min and blocked by PBS solution containing 5% donkey serum and 0.2% Triton X-100 for 1 hour at room temperature. Primary antibodies were applied overnight and secondary antibodies were applied for 2 hours. The following antibodies were used: rabbit anti-Tuj1 (Covance, 1 : 1,000), mouse anti-Tuj1 (Covance, 1 : 1,000), mouse anti-MAP2 (Sigma, 1 : 500), rabbit anti-synaptophysin (Chemicon, 1 : 1,000), rabbit anti-v-Glu (Millipore, 1 : 2,000), and rabbit-anti-GABA (DSHB, 1 : 500). Alexa-488-, Alexa-594- and Cy5-conjugated secondary antibodies were obtained from Invitrogen. 4′,6-Diamidino-2-phenylindole (DAPI) was from Sigma (1 : 10,000).

Cells from SC-DRG co-cultures were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min and then permeabilized in a mixture of 95% ice-cold methanol and 5% acetone at −20 °C for 5 min. Afterwards cells were incubated in blocking solution (2% horse serum, 2% bovine serum albumin (BSA), 0.1% porcine gelantine) for 1 h at room temperature before incubation in primary antibodies (mouse anti-MBP 1:500 (Covance), rabbit anti-TUJ1 1:250 (Covance)) overnight at 4 °C. The next day, cells were washed in PBS for three times and incubated in secondary antibodies (Alexa 488 donkey anti mouse 1:1000 (Invitrogen) and Alexa 568 donkey anti rabbit 1:1000 (Invitrogen) diluted in blocking solution with 0.2 µg/µl 4’,6’-diamidino-2-phenylindole (DAPI; Sigma) for 1 h at room temperature. Following three washing steps in PBS cells were mounted on slides in Mowiol mounting solution (9.6% Mowiol (Sigma), 24% Glycerol, 0.1 M Tris-HCl). Fluorescent images were taken using a Axiophot Observer Z (Zeiss) with a Colibri light source (Zeiss) and MRM camera (Zeiss). Acquisition and processing of the images was carried out using Zen2.6 blue software (Zeiss), FIJI (NIH) and Illustrator 2020 (Adobe).

Neuronal cultures were fixed with 4% paraformaldehyde for 15 min at room temperature and then treated with PBS containing 0.1% Triton X-100. After a 15 min PBS wash, cells were blocked with 5% BSA in PBS for 1 h, then incubated with the primary antibody in PBS at 4 °C overnight and the next day after a few PBS washes with secondary antibodies for 1 hour at ambient temperature. This process was followed by a 10 min incubation with DAPI and a final set of PBS washes. The coverslips were mounted on glass slides using PVA-DABCO. Primary antibodies used were rabbit anti-Tuj1, (1:500, Covance), chicken anti-MAP2 (1:400, Abcam), rabbit anti-αSynuclein (1:500, Invitrogen), rabbit anti-TH (1:500, Pel-Freez), mouse anti-PSD95 (1:500, Life Tech), rabbit anti-Synapsin I (1:500, Calbiochem), anti-Fibronectin (1:200, Sigma). Corresponding Alexa Fluor^TM secondary antibodies were then used (1:1000). For detecting protein aggregates, the PROTEOSTAT® Aggresome Detection kit was used according to the manufacturer’s instructions.
Confocal z-stacks were acquired with a Zeiss LSM 880 Airy scan microscope (Carl Zeiss, Microimaging Inc.) using 405 nm Diode laser, 488 nm Argon, and 543 nm HeNe lasers with a Plan NeoFluar 40×/1.3 oil DIC or a Plan-Apochromat 63×/1.4 oil DIC objective.

For western analysis, 10% brain homogenates were prepared and cleared of nuclei as described [69] (link). The blotting procedure used precast 10% gels with MES buffer (Invitrogen). For figure 1, antibodies were mouse anti-PrP SAF61 1:2000 (Cayman Chemical), mouse anti-GFP 1∶2000 (Roche), Rabbit anti-Tuj1 1:4,000 (Covance), followed by appropriate fluorescent secondary antibodies and scanned on the Odyssey by Li-cor. The blots in figure 5 were similar except mouse anti-PrP SAF83 (1∶2,000) was used.