Irdye 680lt donkey anti rabbit

Spinal cords from wild type and knockout mice (as determined by PCR genotyping) were dounce homogenized in PBS containing protease inhibitors, then sonicated. Lysates were centrifuged at 20,000 g for 30 minutes at 4°C. Protein concentrations were determined by Bradford Assay and normalized. After adding loading dye, proteins were separated by SDS-PAGE, then transferred to a PVDF membrane for 90 minutes at 50 V. The membrane was blocked in Blocking Buffer (Rockland), then incubated at room temperature with PREPL primary antibody (Polyclonal PREPL antibody was generated by Open Biosystems in rabbits using a peptide epitope (EELGLDSTDAFEALKKYLKF) derived from murine PREPL) overnight at 4°C. The membrane was then washed 3× with TBS-T for 5 minutes each, followed by incubation with secondary antibody (IRDye 680LT Donkey Anti-Rabbit (LICOR)) at room temperature for one hour. After washing 3×, the western blot was imaged on a LICOR Odyssey imager.

In vitro differentiated ES cell-derived and LUHMES-derived neurons and whole brain samples were prepared for western blotting as previously described (3 (link)). Extracts were run on TGX 4–20% gradient gels (BioRad) loading extract equivalent to ∼5 × 10⁵ nuclei per well. Samples were run on duplicate gels and transferred to nitrocellulose membrane overnight in the cold at 25 V. Western blots were processed as described previously (3 (link)) using the following antibodies: anti-MeCP2 (N-terminus): mouse monoclonal Men-8 (Sigma), anti-NeuN: rabbit polyclonal ABN78 (Millipore) and anti-histone H3: rabbit polyclonal ab1791 (Abcam). Western blots were developed with IR-dye secondary antibodies (IRDye 800CW donkey anti-mouse, IRDye 680LT donkey anti-rabbit, LI-COR Biosciences) and scanned using a LI-COR Odyssey machine. Images were quantified using Image Studio Lite software (LI-COR Biosciences). The ratio of NeuN: histone H3 signals for each lane was used to check for equal neuronal in vitro differentiation and clones that did not differentiate well were discarded. MeCP2 levels were normalized to the histone H3 signal for each lane to compare the amount of MeCP2/nucleus between samples.

Lysates were prepared as described. 2x Laemmli buffer was added and samples loaded onto polyacrylamide gels. Gels were run at 120 V for 75 min in running buffer (0.25 M Tris, 1.93 M glycine, 0.1% SDS). Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Cressier, Switzerland) in transfer buffer (0.25 M Tris, 1.93 M glycine, 20% methanol) at 350 mA for 45 mins. Membranes were blocked in 5% BSA in TBS for 1 h and incubated with primary antibodies including rabbit anti-CRMP2 (1:1000; Sigma-Aldrich; C2993), rabbit anti-PKCγ (1:1000; Santa Cruz Biotechnology, Santa Cruz, USA; sc-211), rabbit anti-pCRMP2 (1:500; ECM Biosciences; CP2251) and mouse anti-actin (1:2000; Sigma-Aldrich; A5441) overnight at 4 °C. Secondary antibodies IRDye® 800CW Donkey anti-mouse (Li-Cor, Bad-Homburg, Germany; 926-32212) and IRDye® 680LT Donkey anti-rabbit (Li-Cor; 926-68023) were added at a concentration of 1:5000 diluted in TBS-T. After washing, the signal was detected using the Odyssey® Fc Imaging System and software (LI-COR Biosciences, Bad Homburg, Germany). To determine the ratios, pCRMP2/CRMP2 gels were run in parallel for total CRMP2 and pCRMP2. The immunoreactivity in each gel was normalized to the actin signal and the ratio evaluated by (pCRMP2/actin)/(CRMP2/actin) and then normalized to wild-type controls.

The formalin-fixed catheters were prepared as described previously^{25 (link),37 (link)}. Briefly, catheter samples were washed three times with PBS and subsequently blocked with PBS containing 1.5% BSA and 0.1% sodium azide overnight at 4 °C. The blocked catheters were then washed three times with PBS-T and incubated with Enterococcus polyclonal antibody (PA1-73120, Invitrogen, validation statement for immunofluorescence application available at: PA1-73120">https://www.thermofisher.com/antibody/product/Enterococcus-Antibody-Polyclonal/PA1-73120) and E. coli serotype-O/K Polyclonoal Antibody (PA1-73032, Invitrogen, validation statement for immunofluorescence application available at PA1-73032">https://www.thermofisher.com/antibody/product/E-coli-serotype-O-K-Antibody-Polyclonal/PA1-73032) at 1:400 dilutions in dilution buffer (PBS-T, 0.1% w/v BSA, 0.5% methyl alpha-D-mannopyranoside) for two hours at room temperature. The catheters were then washed and incubated with IRDye 680LT Donkey anti-Rabbit (LI-COR, 926-68023) and IRDye 800CW Donkey anti Goat (LI-COR, 926-32214) antibodies (1:10,000 dilution in dilution buffer) for one hour at room temperature. The catheters were then washed three times in PBS-T and imaged using the Odyssey Imaging System (LI-COR Biosciences) and merged in Adobe Photoshop. The control portion of the catheter was not incubated with primary antibody.