Alexa conjugated secondary antibody

Cells were allowed to migrate through the microchannels for at least 3 hours but no longer than 10 hours. Cells were washed 2 times in PBS for 5 minutes each and fixed in either 100% ice cold methanol (for Glu-tubulin and tubulin) or 4% methanol-free paraformaldehyde (all other proteins). Cells were again washed 3 times in PBS for 5 minutes each and blocked for one hour in PBS containing 5% goat serum and 0.3% Triton X-100. Cells were incubated with primary antibodies overnight, diluted in PBS containing 1% bovine serum albumin and 0.3% Triton X-100. The following day cells were washed 3 times in PBS for 5 minutes each and then incubated with Alexa-conjugated secondary antibodies (ThermoFisher Scientific) at 1:500 in PBS containing 1% bovine serum albumin and 0.3% Triton X-100. Cells were washed 3 times again in PBS for 5 minutes and then nuclei were stained with DAPI and imaged.
Western blots were blocked using Intercept TBS blocking buffer (Li-Cor) and incubated with primary antibodies at concentrations listed in the reference table, diluted in blocking buffer with 1% Tween-20 overnight at 4°C. Secondary antibodies conjugated with IRDye (LiCor) were used at 1:10,000 in blocking buffer with 1% Tween-20 for one hour at room temperature. Blots were imaged using a Li-Cor Odyssey imaging system.

Primary neurons and coronal brain sections were washed in PBS containing 0.3% Triton-X100, blocked in the same solution containing 5% BSA, and then incubated in primary antibodies for 1–2 h. They were then washed three times and then incubated with secondary antibodies containing fluorophores for 1 h before three final washes. Primary antibodies included rabbit anti-GABA 1:500 (Sigma A2052), rabbit anti-parvalbumin 1:400 (Swant, PV-27). Alexa-conjugated secondary antibodies (Thermo Fisher) were used to detect primary antibodies. Native tdTomato fluorescence was imaged, and in vitro primary, MGE cultures were also labeled for DAPI using NucBlue Fixed Cell ReadyProbes (Thermo Fisher, R37609). A Nikon eclipse Ts2R microscope (Photometrics CoolSnap dyno camera) and Leica DM2000 microscope (DFC3000G camera) captured the primary culture and transplant images, respectively.

NHCFs were grown in chamber slides (#154534, Thermo Fisher Scientific) and treated with TGF-β1 after silencing the PCAF. The NHCFs were fixed with 3.7% (v/v) paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS. After blocking with 5% BSA at room temperature for 30 min, the cells were incubated with primary antibodies against SMAD2 (1:100; CST) or COL1A1 (1:100; Abcam) in a permeabilization buffer (0.2% Triton X-100, 1% BSA in PBS). Alexa-conjugated secondary antibodies (Thermo Fisher Scientific) were used to confirm primary antibodies. Cells were covered with antifade solution containing 6-diamidion-2-phenylindole (DAPI, #S36939, Thermo Fisher Scientific). The cells were visualized by use of a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany).

For postnatal mouse retina immunostaining, eyes were collected and fixed in 4% PFA in PBS for 20 min at room temperature. After microdissection, retinas were fixed in 4% PFA for an additional 45 min, followed by two PBS washes of 10 min each. Retinas were blocked and permeabilized with PBTS buffer (0.3% Triton X-100, 3% FBS and 3% donkey serum) for 1 h. Samples were then incubated overnight at 4 °C in biotinylated isolectin B4 (diluted 1:50; Vector Laboratories, B-1205) and primary antibodies (Supplementary Table 2) diluted in PBTS buffer. After five washes of 20 min each in PBTS buffer diluted 1:2, samples were incubated for 2h at room temperature with Alexa-conjugated secondary antibodies (Thermo Fisher). After three washes of 30 min each in PBTS buffer (diluted 1:2), and two washes of 10 min each in PBS, retinas were mounted with Fluoromount-G (SouthernBiotech).

BHK or HeLa cells were grown on glass coverslips and infected with sMVA or recombinant sMVAs encoding S and/or N proteins at an MOI of 5 for 6 hours at 37°C in a humidified incubator (5% CO₂). After infection, cells were fixed for 15 min in 2% PFA and then directly permeabilized by addition of ice cold 1:1 acetone/methanol for 5 min on ice. Cells were blocked for 1 hr with 3% BSA at room temperature, incubated with primary antibody mix (1:500) against the S2 subunit or N for 1 hr at 37°C, and then incubated with Alexa-conjugated secondary antibodies (ThermoFischer) (1:2000) for 1 hr at 37°C, with washing (PBS + 0.1 % Tween20) between each step. For detection of cell membranes and nuclei, cells were incubated with Alexa-conjugated wheat germ agglutinin at 5 μg/mL (Thermo Fisher) and DAPI for 10 minutes at room temperature. Coverslips were washed and mounted onto slides with Fluoromount-G (SouthernBiotech). Microscopic analysis was performed using a laser-scanning confocal microscope (Zeiss, LSM700). Images were acquired and processed using Zen software (Zeiss).

Antibodies used were rabbit cC3 (1:250, Catalog #9661S, Cell Signaling Technologies, Danvers, MA, USA), rabbit DsRed (1:500, Catalog #632496, Takara, Mountain View, CA, USA), mouse nAChR α4 Subunit Antibody, clone 369 (1:50, Catalog #MABN1833, MilliporeSigma, St. Louis, MI, USA), rabbit phosphoS6^SER240/244 (1:400, Catalog #5364, Cell Signaling Technologies, Danvers, MA, USA), mouse PSD95 (1:200, Catalog #75-028, NeuroMab, Davis, CA, USA), goat Vesicular Acetylcholine Transporter (VAChT) antibody (1:500, Catalog #ABN100, MilliporeSigma, St. Louis, MI, USA), rabbit Vglut1 (1:500, Catalog #135303, Synaptic Systems, Goettingen, Germany), rabbit VIP (1:250, Catalog #20077, Immunostar, Hudson, NY, USA) and Alexa-conjugated secondary antibodies (1:300, Thermo Fisher, Eugene, OR, USA). Sections were cover-slipped with Vectashield (Vector Labs, Burlingame, CA, USA).

Patient #1 never-cultured pleural effusion or ascites cells were plated at ~3,000 cells/well in 384-well black plates with clear bottom in Renaissance medium (Cellaria) with 5% FBS, 25 ng/ml cholera toxin (Sigma), and 1% antibiotic/antimycotic (Life Technologies); to this was added 20% filtered pleural effusion fluid from another patient. After 3–4 days of culture, cells were washed twice with PBS and fixed in 2% paraformaldehyde (Electron Microscope Sciences) in PBS for 15 min. Cells were washed thrice with PBS and permeabilized in 0.1% Triton X-100 in PBS. Permeabilized cells were incubated with primary antibody in 2% bovine serum albumin overnight at 4 °C. Samples were washed twice with PBS containing 0.05% Tween-20, then incubated with Alexa-conjugated secondary antibodies (Thermo Fisher) and DAPI (Invitrogen) for one hour. Samples were washed twice and stored in PBS at 4 °C. Imaging was performed using an automated high-throughput fluorescence microscope (Olympus scanR). This experiment was performed one time.