Alexa fluor 488 conjugated donkey anti mouse igg secondary antibody

FOP- and resFOP-ML were fixed using 2% paraformaldehyde for 10 min and washed with PBS 3 times. 50–100 μL suspensions containing 50,000–100,000 fixed cells were applied directly to the slide (Matsunami), dried at room temperature, and permeabilized with 100% methanol at 4 °C for 10 min. Samples were blocked with Blocking One or Blocking One-P (Nacalai Tesque) for 60 min and then incubated with anti-CD14, CD16, LYVE-1, or p-Smad5 antibody diluted in Can Get Signal Immunostain Solution B (Toyobo) for 16 to 18 h at 4 °C. Next, the samples were washed 3 times in 0.2% Tween-20 (Sigma-Aldrich) in PBS and incubated with Alexa Fluor 488 conjugated donkey anti-mouse IgG secondary antibody (Abcam) and Alexa Fluor 647 conjugate donkey anti-rabbit IgG secondary antibody (Thermo Fisher Scientific) diluted in Can Get Signal Immunostain Solution B for 1 h at room temperature. DAPI (10 μg/mL) was used to counterstain nuclei.

Slices from electrophysiology were postfixed in 4% paraformaldehyde (PFA) solution for a minimum of 2 d. Slices were washed three times for 10 min each with PBS and placed into blocking solution containing PBST (PBS containing 0.1% Triton X-100) and 5% normal donkey serum. Slices were blocked for 2 h at room temperature. The mouse monoclonal Anti-ChAT antibody (1:1000; Atlas Antibodies; AMAB91130) was added to the blocking solution at a concentration of 1:1000. Tissue was incubated overnight at 4°C with gentle rocking. Slices were washed three times for 10 min each using PBST. Following the third wash, slices were incubated with Alexa Fluor 488-conjugated donkey anti-mouse IgG secondary antibody (1:200; Abcam; ab205718) in PBST (1:200) for 2 h. Slices were washed for three times using PBST under gentle agitation. Brains were mounted onto microscope slides using ProLong Gold Antifade mounting medium (ThermoFisher Scientific) and covered with cover glasses, and sealed with clear nail polish. Images were collected on a Zeiss LSM800 confocal microscope from three different tissue sections per rat with a minimum of three biological replicates. Optical sections were taken from a range of 5–15 μm from the surface of the brain tissue. All images were acquired using the same acquisition parameters, including laser power, gain, and offset.