Rabbit anti rat immunoglobulins hrp

The formalin‐fixed paraffin‐embedded lung tissue sections (4.25 μm in thickness) were stained with hematoxylin and eosin (H&E). An immunohistochemistry assay was employed using several primary antibodies against SARS‐CoV‐2 spike protein (mouse, GTX632604, GeneTex), SARS‐CoV‐2 nucleocapsid protein (rabbit, GTX635679, GeneTex), CD31 (rabbit, ab28364, Abcam), CD41 (rabbit, ab134131, Abcam), CD45 (rat, #103202, Biolegend), MPO (rabbit, ab9535, Abcam), CD68 (rabbit, ab125212, Abcam), and P‐selectin/CD62P (rabbit, ab255822, Abcam). The used secondary antibodies include Goat Anti‐Rabbit Immunoglobulins/HRP (P0448, Dako), Goat Anti‐Mouse Immunoglobulins/HRP (P0447, Dako), and Rabbit Anti‐Rat Immunoglobulins/HRP (P0450, Dako). The liquid DAB⁺ Substrate Chromogen System (K3468, Dako) was used for color development. DAB signal‐positive areas of CD41, CD45, MPO, and CD68 were quantified using an ImageJ software (NIH). The area with a particularly high (N) protein positive signal was defined as “N protein signal‐rich area,” and the positive areas of CD41, CD45, MPO, and CD68 were quantified using an ImageJ software (NIH).

Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections using following antibodies and dilution ratios: rat monoclonal anti-mouse F4/80 antibody (1:250, Abcam, Cambridge, UK), rabbit polyclonal anti-mouse CD3 antibody (1:500, Abcam), and rat monoclonal anti-mouse E-cadherin (1:200, eBioscience). Briefly, after dewaxing and rehydration, a heat-inducing antigen retrieval procedure using citrate buffer at pH 6.0 for 21 min was performed on all tissue sections, with subsequent washing in PBS and endogenous peroxidase blocking with EnVisionTM FLEX Peroxidase-Blocking Reagent (Dako, Agilent, Santa Clara, CA, USA) for 10 min. Sections were incubated with primary antibodies overnight. Sections stained with anti-F4/80 and anti-E-cadherin antibody were incubated with secondary polyclonal rabbit anti-rat immunoglobulins/HRP (1:500, Dako) for 60 min. The CD3 sections were treated by applying the commercial EnVisionTM FLEX/HRP detection reagent (Dako). Immunoreactions were developed with diaminobenzidine (DAB, Dako) diluted in EnVisionTM FLEX Substarte Buffer (Dako). The sections were counterstained with haematoxylin.