Alexa fluor 647 conjugated goat anti mouse antibody

Twenty-four hours after seeding, the SK-OV-3 cells were grown in a cell incubator (37 °C, 5% CO₂) in the RPMI-1640 medium supplemented with 20 μM nocodazole for four h. To make repolimerization of microtubules possible, the nocodazole-containing medium was removed. Then, the cells were placed in the standard medium and grown in a cell incubator for 10 min. Afterward, the cells (including their cytoskeleton) were fixed with methanol at −20 °C for 20 min, followed by their immunofluorescence staining with the alpha tubulin-specific primary mouse antibody (Thermo) and the secondary Alexa Fluor 647-conjugated goat anti-mouse antibody (Thermo), according to the protocol described by Grzybowska et al. [66 (link)]. Visualization of the cells was performed under the LSM8 microscope with the 63× objective.

Transfected cells were fixed in 4% paraformaldehyde and then incubated for 30 min at RT with an in-house RVG protein specific monoclonal antibody (diluted 1:1000 in PBS) [22 (link)]. Cells were washed with PBS, followed by 30 min incubation at RT with an AlexaFluor647^® conjugated goat anti-mouse antibody (Thermo Fischer Scientific, Waltham, MA, USA) diluted 1:000 in PBS). Cells were washed and resuspended in PBS for flow cytometry analysis using a BD FACS Canto flow cytometer (BD Biosciences, San Jose, CA, USA).

Clodronate liposomes (CL lipo) (Yeasen, Shanghai, China) were used for macrophage depletion in chickens as reported previously [21 (link)]. Six 5-week-old SPF chickens were randomly divided into two groups, with three chickens in each group. Three 5-week-old SPF chickens were intravenously (i.v.) treated with 250 μL of CL lipo or the control reagent, PBS liposomes (PBS lipo), respectively. The efficiency of macrophage depletion was verified using IFA in the spleen on day 5 post-treatment (pt). The spleens were fixed with 10% buffered formalin and prepared into tissue sections. A mouse anti-chicken KUL01 monoclonal antibody (1:50) was added to the sections as the primary antibody and incubated at 4 °C overnight. After washing with PBS 3 times, the sections were then incubated with Alexa Fluor 647-conjugated goat anti-mouse antibody as the secondary antibody (1:200) (ThermoFisher Scientific) at 37 °C for 1 h. After washing with PBS, the cell nucleus was stained with DAPI. The images were captured using a fluorescence microscope (Leica).

Flow cytometry fluorescence-activated cell sorting (FACS) was applied to detect the presence of α_Vβ₆ integrin in FLSs. FLSs (n = 4) were detached by scraping and harvested on ice-cold PBS containing 1 mM MgCl₂ and 0.1% BSA; 4 × 10⁵ cells were used for each condition. The cell scraper was used to prevent ablation induced by trypsin, and the experiment was performed on ice to prevent integrin internalization^{25 (link)}. U87 glioblastoma cells that naturally expressed α_Vβ₆ integrin were used as a positive control^{26 (link)}. Primary antibody recognizing α_Vβ₆ integrin (Merck; Darmstadt, Germany) was used at a concentration of 100 µg/mL in PBS/MgCl₂/BSA. After incubation for 40 min on ice and two washes with PBS/MgCl₂/BSA, the cells were incubated for 20 min on ice in the dark with Alexa Fluor 647-conjugated goat anti-mouse antibody (Thermo Fisher Scientific). After two washes, the cells were resuspended in 500 µL of PBS/MgCl₂/BSA and filtered with a 100 µm strainer (Sysmex; Norderstedt, Germany) in FACS tubes; 1 µL of DAPI dye (Thermo Fisher Scientific) was added to identify dead cells. Flow cytometric measurements were performed with a FACSCanto^TM II (BD Biosciences; San Jose, CA, USA), and 10⁴ cells were analyzed in each assay. Flow cytometry standard files were then analyzed by FlowJo^TM software (BD Biosciences).