Axio observer 7 fluorescence microscope

To study HL-60 adhesion to endothelial cells, HULEC cells were seeded in 24-well cell imaging plates (Merck Millipore, Darmstadt, Germany) and treated as described above. After washing with PBS, carboxyfluorescin succinimidyl ester (CFSE)-labeled HL-60 cells (4.5 × 10⁴) were added at 300 µL of FBS-free medium. Cells were co-incubated for 30 min at 37 °C, 5% CO₂ and then nonadhered HL-60 cells were removed and plates were rinsed twice with 1 mL FBS-free medium. Then, HL-60 cell adhesion to endothelial monolayers was measured as an area of residual green fluorescence staining using an Axio Observer 7 fluorescence microscope and ZEN software v.3.3 blue edition (Carl Zeiss Inc., Dresden, Germany).

Following fixation with 4% paraformaldehyde for 10 min, cells were washed 3 times for 5 min with 0.1 M phosphate buffer (0.1 M Na₂HPO₄ × 2H₂O, 0.1 M NaH₂PO₄ × H₂O, pH 7.4) and blocked with 3% bovine serum albumin and 0.1% Triton X-100 in 0.1 M phosphate buffer for 30 min. After incubation at 4 °C overnight with 1:50 rabbit α-galectin-1 (Abcam, Cambridge, UK), 1:100 mouse α-smooth muscle-α-actin (Santa Cruz, Dallas, TX, USA), 1:100 mouse α-N-cadherin (Thermo Fisher), 1:100 mouse α-integrin-β1 (Merck), 1:100 mouse α-cytokeratin-8 (Merck), 1:100 phalloidin Alexa Fluor 555 (Thermo Fisher) in 0.3% bovine serum albumin, and 0.01% Triton X-100 in 0.1 M phosphate buffer, specimens were washed again 3 times for 10 min each with 0.1 M phosphate buffer and incubated with 1:1000 goat anti-rabbit Alexa Fluor 488 or goat anti-mouse Alexa Fluor 488 (both from Thermo Fisher) in 1:10 diluted blocking solution for 1 h at room temperature. After nuclear staining with Hoechst 33342 (Thermo Fisher), specimens were washed again 3 times with 0.1 M phosphate buffer and mounted with the ProLong glass antifade mounting medium (Thermo Fisher). Immunofluorescence staining was analyzed by an Axio Observer 7 fluorescence microscope with an Apotome 2 module (Zeiss) and documented using the ZEN software Blue edition 3.0 (Zeiss).

Mice was perfused with 4% PFA and the cervical spinal cord was dissected. Next, the tissues were fixed in 4% PFA overnight at 4°C, followed by processing in 20% sucrose, embedded in OCT, and then sectioned into 20 μm, and subjected to tissue staining after cryosection. A blocking solution was prepared with 0.1% Tritox-100, 3% goat serum, 0.1% BSA plus PBS solution, and blocked at room temperature for 1 h (Xiang et al., 2020 (link)). After blocking, sections were incubated overnight at RT with the specific primary antibodies (anti-CXCR3, 1:5,000, 26756-1-AP, Proteintech; anti-NeuN, 1:200, Millipore, MAB377; anti-GFAP, 1:500, Millipore, MAB360; anti-CD11b, 1:200, Abcam, ab8878). Then, they were washed 3 times in PBS, and incubated with the appropriate secondary antibodies (Alex Fluor™ 647 goat anti-rabbit IgG (H + L), 1:1,000, Thermo Fisher Scientific, #1851447; Alex Fluor™ 568 goat anti-mouse IgG (H + L), 1:300, Thermo Fisher Scientific, 1862187) for 1 h at RT. After incubated with 5 μg DAPI, tissue sections were washed, mounted and then imaged after drying. All imaging were performed on a Zeiss Axio Observer 7 Fluorescence Microscope. The immune signal intensity was quantified in a semi-automatic manner using Image-Pro Plus software and 4–5 images were counted for each mouse as described previously (Zhao et al., 2013 (link); Xiang et al., 2020 (link)).