Widefield epifluorescence ti2 microscope

Two 10-week-old female C57BL/6NRj mice were sacrificed using isoflurane followed by cervical dislocation. Organs were collected and put into 10% formalin. The tissues were dehydrated and embedded in paraffin and 4-μm sections were generated from the paraffin blocks. The sections were heated on the slides at 65 °C for 30 min and then processed for antigen retrieval in EnVision™ FLEX Target Retrieval Solution, High pH (Tris/EDTA buffer, pH 9) for 20 min at 97 °C. The sections were washed for 5 min in PBST followed by protein block (Agilent Technologies, Santa Clara, CA, USA) for 5 min. After washing with PBST for an additional 5 min, 20 μg/ml anti-HS (Amsbio) coupled to Alexa Flour 488 and 20 μg/ml rabbit anti-human urinary A1M (K:322 IgG, prepared in-house by immunization with human urinary A1M purified as described [47 (link)]), coupled to Alexa Fluor 647, were added and incubated at 4 °C overnight. The sections were washed for 5 min with PBST followed by incubation with Hoechst (Thermo Fisher Scientific) diluted 1:2000 for 15 min. After washing for 5 min with PBST, cover glasses were mounted using ProLong Gold antifade (Thermo Fisher Scientific). Microscopy was performed on a Widefield Epifluorescence Ti2 microscope equipped with a Nikon DS-Qi2 camera.

Lung tissue sections were fixed as reported above. Lung samples underwent antigen retrieval (pH 9 buffer) using a Dako PT Link pre-treatment module (Agilent). Samples were washed and blocked for 10 min (Dako protein block; Agilent, Santa Clara, CA) before being treated with primary antibodies overnight. Mouse anti-COL1A1, ARG1, and rabbit anti-FDPS, CD206 (Abcam, Cambridge, United Kingdom) antibodies were used. Alexa Fluor 488-conjugated goat/anti-mouse and Alexa Fluor 647 goat/anti-rabbit (Invitrogen, Waltham, CA, United States) were used as secondary antibodies. Glass cover slips were placed onto slides and mounted with DAPI-containing fluoroshield (Abcam). Microscopy was performed on a Widefield Epifluorescence Ti2 microscope equipped with a Nikon DS-Qi2 camera and fluorescence was quantified using ImageJ software.