Anti mpo antibody

ELF-PEMF-exposed neutrophils suspension (150 μL, 3 × 10⁵ cells/mL) were seeded in sterile poly-L-lysine-coated chamber slides. After 4 h of incubation, neutrophils were fixed with 4% formaldehyde for 30 min and incubated with 1% Triton-X-100 for 5 min at room temperature. Fixed neutrophils were gently washed 3 times with PBS, incubated with 5% BSA solution for 1 h, and then incubated with the primary antibody overnight at 4 °C. Anti-MPO antibody (1:200 v/v, Cat. #sc-52707, Santa Cruz Biotechnology, Heidelberg, Germany) was used as the primary antibody. After the overnight incubation, neutrophils were gently washed 3 times with PBS and incubated with Alexa Fluor 488 goat anti-mouse IgG (1:1000 v/v, Cat. #A10667, Invitrogen, Heidelberg, Germany) for 2 h at room temperature. After gently washing 3 times, neutrophils were incubated with the 2 µg/mL Hoechst 33342 for 20 min to visualize the DNA. A fluorescence microscope was used to capture the images (EVOS FL, Life Technologies, Darmstadt, Germany). The ratio of NETosed cells was obtained by dividing the number of MPO⁺ cells by the number of Hoechst 33342⁺ cells. The analysis was performed as previously described [69 (link)].

Immunohistochemical analysis was performed as already described [50 (link)]. Subsequently, the sections were incubated overnight with an anti-Bromodeoxyuridine (BrdU) antibody (1:100; Santa Cruz Biotechnology) or anti-MPO antibody (1:250; Santa Cruz Biotechnology) or anti-NRLP3 antibody (1:250; Santa Cruz Biotechnology) or anti-COX-2 antibody (1:250; Santa Cruz Biotechnology) or anti-Iba-1 antibody (1:250; Santa Cruz Biotechnology) or anti-GFAP antibody (1:450; Santa Cruz Biotechnology). Sections were washed with PBS and incubated with peroxidase-conjugated bovine anti-mouse IgG, secondary antibody (1:2000 Jackson Immuno Research, West Grove, PA, USA). Specific labeling was provided with a biotin-conjugated goat anti-mouse IgG and avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Images were collected using a Leica DM6 microscope associated with Leica LAS X Navigator software. The number of positive cells was counted in three sections per animal and presented as the number of positive cells per high-power field.

Rats were anesthetized and transcardially perfused with PBS, followed by 0.1 M PBS containing 4% PFA (pH = 7.4). Perfusion-fixed brains were postfixed in paraformaldehyde overnight, followed by dehydration in 40% sucrose. Coronal brain sections (10 µm thick) were cut on a cryostat (CM1950; Leica Microsystems, Wetzlar, Germany), and the sections were blocked for 2 h in 5% bovine serum albumin in PBS with 0.3% Triton X-100. The sections were then incubated overnight at 4 °C in 3% bovine serum albumin in 0.1% Triton X-100/PBS with the following primary antibodies: anti-p47^phox antibody (1:200; sc-14015)and anti-MPO antibody (1:100; sc-34159) (both were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being rinsed three times with PBS, sections were incubated for 2 h in fluorochrome conjugated secondary antibody. After being rinsed with PBS, the sections were examined under a laser scanning confocal microscope (Carl Zeiss LSM 700, Oberkochen, Germany).