Zen 2011 black edition

Immunostaining for KIM-1 and all ENaC subunits were carried out on frozen sections (14 μm). Tissue sections were blocked with 10% goat serum and incubated with primary antibodies: rat anti-KIM-1 (cat. # MAB1817, R&D systems); rabbit anti-α-ENaC and rabbit anti-γ-ENaC [28 (link)]; rabbit anti-β-ENaC [27 (link)], or rabbit anti-NCC (cat. No. ab3553; Abcam). Sections were then incubated with the corresponding fluorescently tagged secondary antibody (AlexaFluor-488, Thermo Fisher Scientific), counterstained with DAPI, and mounted in Prolong Gold Antifade reagent (Invitrogen). Stained samples were imaged using an LSM 800 confocal microscope using Zen 2011 (Black Edition) version 8.1.5.484 (Carl Zeiss Microscopy, Jena, Germany). Image analysis was conducted using Adobe image suite software.

Viral antigen detection in SARS-CoV-2 animal models was accomplished by immunofluorescence staining. The lung was fixed with 4% paraformaldehyde, paraffin embedded and cut into 3-μm sections. Slides were deparaffinized and rehydrated, then incubated with PBS/0.02% Triton X-100 and blocked with 5% BSA at room temperature for 1 h. The anti-SARS-CoV-2 N protein antibody was added to the sections, followed by washing and incubation with Alexa Fluor 568 goat-anti-human IgG (Invitrogen) at 1:200 dilution. After washing in PBS, slides were stained with DAPI (Invitrogen) at 1:100 dilution. The images were acquired using a ZEN 2011 Black Edition (Carl Zeiss MicroImaging GmbH) and LSM 700 confocal microscopy (Carl Zeiss AG).

Plasticity of dendritic spines was analyzed as described before [26 (link),27 (link),28 (link)]. In total, 7–10 dendrites per animal with a length of ~15–60 µm were obtained for analysis. Spines of all shapes with a length of >0.4 µm were counted manually using Zen 2011 Black Edition (Zeiss, Oberkochen, Germany). Due to resolution limitations, spines emerging laterally from the shaft (xy-plane) but not from the z-plane were taken into consideration. Spines that did not change their position (<0.5 µm) on the dendritic segment between two imaging time points (one week) were counted as stable. Spines were counted as lost when their length was less than 0.4 µm and gained when their length was greater than 0.4 µm. In current study, mice were imaged once per week; therefore, we counted spines as transient when they appeared at one imaging time point (>0.4 µm length) but were lost at the next time point (<0.4 µm length) one week later, as already used previously [17 (link)]. The spine turnover rate was calculated as follows: (N_gained + N_lost)/(2 × N_total)/I_T. N_gained represents the number of newly gained spines at one time point, N_lost is the number of depleted spines, N_total is the number of all spines at one time point, and I_T is the days between two imaging sessions [26 (link),28 (link)].