Sc 20060

Frozen tissue sections were fixed by dropping 4% PFA and allowing to stand for 15 min. It was then washed by immersing it in 10 mM glycine/PBS three times for 5 min. A 2% gelatin solution was added dropwise to the tissue section and allowed to stand for 20 min for blocking. The washing was repeated 3 times by immersing in 10 mM glycine/PBS for 5 min. After further immersing in 0.1% BSA/PBS for 5 min, 1% BSA/PBS containing an antibody against CD68 (1:50, sc-20060, Santa Cruz) was added dropwise, and the mixture was allowed to stand for 40 min. The washing was repeated 5 times by immersing in 0.1% BSA/PBS for 5 min. Next, 1% BSA/PBS containing goat anti-mouse IgG H&L labelled with Alexa Fluor 488 (Ex: 496 nm, Em: 519 nm) (1:200, ab150113, Abcam) was added dropwise and allowed to stand for 40 min. The washing was repeated 5 times by immersing in 0.1% BSA/PBS for 5 min. Counterstaining was performed by immersing in a hematoxylin solution for 30 s and rinsing with a running water for 5 min. Moisture around the section was wiped off with a Kimwipe, dried for about 1 min, then the encapsulant New MX was added dropwise, and the mixture was sealed with a cover glass. The sample was observed with an inverted confocal microscope.

Following electrophysiological measurement, the atrial tissues were quickly removed and fixed in 4% paraformaldehyde, embedded in paraffin then sectioned to 5 microns. To identify KCa3.1 and the pro-inflammatory M1 macrophages, the primary KCa3.1 antibody (bs-6675R, Bioss, 1:200) and CD68 antibody (KP1) (sc-20060, Santa Cruz, 1:50) was added to the sections overnight at 4°C. Then, the sections were incubated with the secondary antibody Fluorescein (FITC)–conjugated Affinipure Goat Anti-rabbit IgG (Aspen, AS1110; 1:50) and Fluorescein (CY3)–conjugated Affinipure Goat Anti-mouse IgG (Aspen, AS1111; 1:50) and cell nuclei by DAPI. The results were analyzed using Image-Pro Plus 6.0 software. In each group, four atrial samples from different individuals were randomly selected and used to calculate the mean number of macrophage and quantificat KCa3.1 expression in macrophage in three different fields at 400x magnification. The mean optical density of KCa3.1 in macrophage = Sum of KCa3.1 optical density values in CD68 marked area/CD68 marked area.

Immunofluorescence (IF) was used to observe the polarization of M1/M2 macrophages in the bovine bone mineral grafting area with mechanical force-induced bone remodeling during OTM. The alveolar bone samples from the C, O, and O + B groups (including teeth) were fixed in 4% paraformaldehyde for 48. Then, the samples were decalcified using ethylenediaminetetraacetic acid (EDTA), followed by dehydration in gradient alcohol and clearing in xylene. After embedding in paraffin, serial Sects. (5 μm thick) were acquired, mounted on slides, and stained using IF. To detect macrophage polarization, antibodies recognizing CD11b (integrin subunit alpha M) (1:1000, ab133357, Abcam, Cambridge, UK) as a cell surface marker for M0 macrophages were used. Antibodies recognizing CD68 (1:50, sc-20060, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used to label the M1 macrophages (CD68⁺CD11b⁺). Antibodies recognizing CD206 (1:10,000, 60,143-1-Ig, Proteintech, Rosemont, IL, USA) were used to label the M2 macrophages (CD206⁺CD11b⁺). The M1/M2 polarization of macrophages in the OTM area with bovine bone mineral grafts was observed [18 (link)]. Five random fields of view were analyzed using Image J software (NIH, Bethesda, MD, USA).

Immunohistochemical and immunofluorescence staining were carried out according to the procedure described in our previous study (32 (link), 33 (link)). FFPE sections (3 μm) were rehydrated and incubated with primary antibodies against α-SMA (1:100, ab5694, Abcam), Twist (1:100, ab175430, Abcam), HDAC6 (1:100, A11259, ABclonal), CD163 (1:100, GB11340-1, Servicebio), Collagen I (1:400, GB11022-3, Servicebio), CD68 (1:100, sc-20060, Santa Cruz Biotechnology) and Arginase-1 (1:100, #93668, Cell Signaling Technology), and then secondary antibodies (Invitrogen). Slides were captured with a Nikon Eclipse 80i microscope equipped with a digital camera (DS-Ri1, Nikon, Shanghai, China).