Alexa fluor 594 conjugated goat anti rabbit igg

Blood obtained from mice was diluted to 1:40 in PBS and washed three times. Blood smears were fixed on glass slides with ethanol/methanol (1:1) for 1 min at – 20°C. Later, the fixed smears were blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature (RT). Mouse anti-B. microti P32 (rBmP32) and rabbit anti-B. rodhaini P26 (rBrP26) polyclonal antisera were applied as the primary antibody on the fixed smears and incubated at 37°C for 1 h in a moist chamber. After washing three times with PBS and rinsing with distilled water, Alexa Fluor^® 594-conjugated goat anti-rabbit IgG or Alexa Fluor^® 488-conjugated anti-mouse IgG (Thermo Fisher Scientific, Massachusetts, USA) was applied as the secondary antibody (diluted 1:200 in 3% BSA in PBS) on the smears, which were incubated at 37°C for 30 min. The slides were then washed three times and incubated with 200 µg/mL Hoechst 33342 solution (Thermo Fisher Scientific) diluted in 3% BSA in PBS containing 50 mg/mL RNase (Qiagen, Hilden, Germany) at 37°C for 10 min. After washing with PBS twice, the glass slides were mounted by adding 10 mL of a 50% glycerol–PBS (v/v) solution and covered with a glass coverslip. The slides were examined and micrographs were taken using the All-in-One BZ-9000 microscope (Keyence, Illinois, USA).

We added rWRS to the wells of an eight-part slide culture of HUVECs at 5 µg/mL in serum-free HuMedia-EG2 medium. After 12 h of incubation at 37 °C, the cells were fixed with paraformaldehyde. After washing the cells with PBS and then blocking them with 1% fetal calf serum, they were treated with monoclonal anti-ICAM-1 antibodies (1:200; Abcam, Tokyo, Japan) and anti-VCAM-1 antibodies (1:200; Abcam) overnight at 4 °C. The cells were washed with PBS and then treated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000; Thermo Fisher Scientific), followed by the staining of the nuclei with DAPI (1:1000; Thermo Fisher Scientific). We analyzed the treated cells using fluorescence microscopy (KEYENCE, Osaka, Japan) [61 (link)]. The fluorescence intensity was measured using the image processing software ImageJ.

RAW264.7 cells were pretreated with MG-132 (5 μM) for 1 h and treated with 2 μM purified EST12 protein for indicated time. Media was removed from stimulated macrophages and the dishes were submerged in 4% paraformaldehyde (PFA). The fixed dishes were submerged in PBS to remove residual PFA and then cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature (RT), washed in PBS and blocked in PBS containing 10% donkey serum. Primary antibodies against FBW7 (28424-1-AP, Proteintech, China) and Myc (M4439, Sigma-Aldrich, USA) were used at a 1:50 dilution in PBS with 3% serum and incubated at 4°C overnight. Then Alexa Fluor 594-conjugated goat anti-rabbit IgG (A-11037, ThermoFisher, USA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (A28175, ThermoFisher, USA) was used at 1:500 dilution in PBS with 3% FBS and incubated at RT for 1 h. Dishes were washed to remove the unbound secondary antibody and the cellular nuclei DNA was stained with DAPI. Fluormount-G Anti-Fade Mounting Medium (Southern Biotech) was added to each well to protect the fluorescent signal. Confocal fluorescence microscopy was performed using ZEISS LSM880 microscope and representative images were captured.

HCT-116 or SW620 stable cells were grown in 4-well chamber slides (Thermo Fisher Scientific) and allowed to attach for 1 day. Cells were fixed and permeabilized with 100% methanol (prechilled at − 20 °C) at room temperature for 5 min and blocked with 1% bovine serum albumin for 1 h. Then, the cells were incubated with anti-FLAG M2, anti-BiP (Abcam), or anti-giantin (Abcam) antibodies. After washing cells with phosphate-buffered saline (PBS), they were incubated with secondary Alexa Fluor® 488-conjugated rabbit anti-mouse IgG or Alexa Fluor® 594-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific) antibodies. Nucleic acids were stained with 4′,6-diamidino-2-phenylindole (Vector Laboratories). Images were acquired using a confocal laser-scanning microscope and analyzed using an LSM image examiner (Carl Zeiss).

For the detection of TNALP and ENPP1, dewaxed paraffin sections were incubated with 1% BSA-PBS and then with rabbit polyclonal antibodies against TNALP at a dilution of 1:300 (1% BSA-PBS). Subsequently, they were incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at a dilution of 1:100 (1% BSA-PBS) for 1 h and then with goat polyclonal anti-ENPP1 at a dilution of 1:200 (1% BSA-PBS) after washing with PBS. Additionally, the sections were incubated with Alexa Fluor 488-conjugated donkey anti-goat IgG (Thermo Fisher Scientific, Inc.) at a dilution of 1:100 (1% BSA-PBS) for 1 h. After embedding the sections using VECTASHIELD hard-set mounting medium with DAPI (Vector Laboratories, Inc. Burlingame, CA, USA), the sections were observed under a light microscope.

Each of control, Actb-knockout and Srf-overexpressing NPCs were seeded on a gelatin-coated culture plate. Cells were fixed with 4% paraformaldehyde (Nacalai Tesque) and permeabilized with 0.5% TritonX-100 in PBS. These cells were treated with Blocking One (Nacalai Tesque) and reacted with Rabbit anti-Mkl1 antibody (Abcam, 49311) at a 1:200 dilution overnight. Specimens were subsequently reacted with Alexa Fluor 594-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, A-11012) at a 1:500 dilution and mounted with VECTASHIELD Mounting Medium (Vector Laboratories) followed by observation using an inverted IX73 fluorescence microscope (Olympus).