Densitoquant software quantcenter

The fresh spleens were collected and fixed in 4% neutral buffered formalin for IHC staining. In brief, tissue sections were incubated with the specific primary antibodies (anti-cap or anti-TLR4 antibodies) at 37°C for 1 h. After washing in PBS, the sections were incubated with a horseradish peroxidase labeled anti-mouse antibody at 37°C for 1 h. Next, freshly prepared DAB was added into the sections at RT for 5 min. Finally, after hematoxylin staining for 1 min, the sections were dehydrated and mounted by neutral gum, and then examined by an optical microscope. Images were captured with a Pannoramic viewer (Pannoramic MIDI, 3D HISTECH), and data were analyzed using DensitoQuant software (QuantCenter, 3DHISTECH). A histochemistry score (H-score) was calculated as previously described (Sun et al., 2019 (link)).

At the end of the experiments, mice were euthanized. Lung and spleen tissues were taken from each mouse. Approximately 75% of the lung tissue was stored at −80°C for the subsequent experiments, and the other 25% of the lung tissue was fixed in 4% formaldehyde for hematoxylin-eosin staining (H&E) according to standard protocols as described previously (26 (link), 27 (link)) with some modifications. Briefly, lung tissue was fixed in 10-fold volume of 4% formaldehyde for 48 h. Next, samples were embedded in paraffin and cut into 4-μm-thick sections. One section from each tissue sample was stained with H&E.
For immunohistochemical staining, spleen tissues were incubated with a monoclonal antibody for TLR4 (Abcam, UK) and then incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. Subsequently, the HRP conjugates were visualized using a diaminobenzidine solution. Images were captured with a Pannoramic viewer (Pannoramic MIDI, 3D HISTECH), and data were analyzed using DensitoQuant software (QuantCenter, 3DHISTECH). A histochemistry score (H-score) was calculated according to a previously reported equation (28 (link), 29 (link)).