Immunocruz rabbit abc staining system

Anti-Olfr68 (Abcam, Cambridge, MA, catalog #ab62606), anti-Olfr1508 (Abcam, catalog #ab65734), and anti-adenylyl cyclase 3 (Santa Cruz Biotechnology, catalog #sc-588) antibodies were used at a dilution of 1:100 on formalin-fixed, paraffin-embedded tissue sections. After deparaffinization, endogenous peroxidase activity was inactivated with 3% hydrogen peroxide for 10 minutes. Normal goat serum was added as blocking agent for 1 hour, followed by an overnight incubation at 4°C with primary antibody and a 30-minute incubation with biotinylated goat anti-rabbit antibody provided with the ImmunoCruz rabbit ABC Staining System (Santa Cruz Biotechnology, catalog #sc2018). Slides were counterstained with hematoxylin.

Formalin-fixed and paraffin-embedded lung autopsy specimens from patients who died of influenza or COVID-19 (N = 2 patients per group) were obtained from the Pathology Department of the INER. Sections of 3–5 μm were processed for hematoxylin–eosin (H&E) staining for histopathological analysis. For immunohistochemistry (IHQ), lung sections were mounted on silane-covered slides, deparaffinized, and the endogenous peroxidase blocked. Sections were incubated overnight at room temperature with an optimal dilution (1:100) of the Mouse Anti-Human CXCL17/VCC-1 Monoclonal Antibody (Clone # 422208, MAB4207, R&D Systems, Minneapolis, MN). Secondary biotinylated antibodies labeled with peroxidase were added, and those attached were revealed with diaminobenzidine (ImmunoCruz™ rabbit ABC Staining System, Santa Cruz Biotechnology, Santa Cruz, CA). Slides were counter-stained with hematoxylin.

Muscle fiber type and Pax7-positive nuclei were determined using the ImmunoCruz^® rabbit ABC Staining System (sc-2018; Santa Cruz Biotechnology, Dallas, TX, USA) according to the manufacturer’s guidelines. Briefly, 8 μm frozen muscle sections were preincubated for 5 min in H₂O₂ and washed twice for 5 min in PBS and blocked for 1 h at room temperature. Subsequently, the sections were incubated overnight at 4 °C with primary antibodies for type 1 muscle fiber (1:500; PAD418Mu02; Cloud-Clone Corp, Katy, TX, USA), type 2A muscle fiber (1:500; PAA755Mu01; Cloud-Clone Corp), type 2B muscle fiber (1:500; PAD416Mu01; Cloud-Clone Corp), and Pax7 (1:100; AP10488B; Abcepta, San Diego, CA, USA). On the next day, sections were washed three times for 5 min in PBS and incubated with biotinylated secondary antibody for 1.5 h. After washing in PBS, the sections were incubated for 30 min with AB enzyme reagent. Sections were then washed and incubated in three drops of peroxidase substrate for 5 min. Finally, sections were washed in deionized H₂O for 5 min, dipped in 90/95/99.5% ethanol and xylene, and mounted with coverslips using mounting reagent (NEW M·X, Tokyo Garasu Kikai, Tokyo, Japan). Tissue slides were observed using PALM MicroBeam IV (ZEISS). The fiber-type distribution and number of Pax7-positive nuclei per muscle fiber was calculated from 200 fibers using the ImageJ software.

Gastric tissues from WT and L22P mice were collected and fixed in 3.7% PFA overnight before paraffin embedding. Tissues were then sectioned along the longitudinal axis for immune-histological staining using ImmunoCruz rabbit ABC Staining System (Santa Cruz, sc-2018). Primary antibodies applied include STING (1:200, Cat. 13647S, Cell Signaling), IRF3 (1:200, Cat. 4302S, Cell Signaling), and p-IRF3 (1:100, Cat. 4947S, Cell Signaling). Stained slides were then scanned using Scanscope (Leica Biosystem). For each slide, 5 fields were randomly selected, and the number of positively stained nuclei and total nuclei were counted.