Anti mpo

Briefly, the formalin-fixed paraffin-embedded tissue sections were deparaffinized, rehydrated, and were subjected to antigen retrieval in EnVision FLEX Target Retrieval High pH Solution (Tris/EDTA, pH9.0; Agilent) at 95 °C for 20 min using PT Link (Agilent Technologies, Santa Clara, CA, USA). The sections were then permeabilized with 0.1% Triton X-100 in PBS for 15 min and blocked with 5% V/V donkey serum (Jackson ImmunoResearch, 017-000-121). The primary antibodies, anti-MPO (R&D SYSTEMS, AF3667) and anti-Bcl-xL (Abcam, ab32370), were added for overnight incubation at 4 °C. The primary antibodies were washed with washing solution 0.05% Tween-20 in PBS followed by incubation with secondary antibodies, Alexa Fluor 488-conjugated Donkey anti-Goat (Jackson ImmunoResearch, 705-545-003) and Cy3-conjugated Donkey anti-Rabbit (Jackson ImmunoResearch, 711-165-152) diluted in donkey serum, for 1 h at room temperature. The secondary antibodies were washed and counterstained with DAPI for nuclear inspection. The slides were scanned using Axioscan7 (Carl Zeiss SA, Oberkochen, Germany) and double-positive cells were analyzed using QuPath’s (v 0.3.2) classifier.

Lung sections were stained using a modified protocol based on published reports (34 (link), 35 (link)). Five micrometer sections from paraffin-embedded lung biopsies from control or IPF patients were dewaxed prior to heat-induced epitope retrieval with Tris-EDTA buffer, pH 9.0. Sections were blocked with Fc block (BD biosciences, UK) before incubation with a blocking buffer (5% goat serum/2.5% BSA/PBS/0.1% Tween-20) for 1 h. Slides were then washed and incubated with anti-citrullinated histone H3 (Abcam, UK) and anti-MPO (R&D systems, UK) antibodies diluted in 0.5x blocking buffer overnight at 4° C. Anti-rabbit Alexa Fluor^® 647-conjugated and anti-mouse Alexa Fluor^® 555-conjugated secondary antibodies (Invitrogen, UK) and DAPI (Sigma, UK) diluted in 0.5x blocking buffer were then added for 30 min. Stained sections were washed, mounted, sealed and visualized using an Olympus inverted fluorescence confocal microscope and analyzed using Fluoviewer software (Olympus).

To prepare protein extracts for western blotting, infected footpads and spleen tissues harvested from each group of mice were homogenized on ice in RIPA buffer (Sigma-aldrich, R0278) added with 1× protease inhibitor cocktails (Thermo, 78410) and 1 mM phenylmethylsuphonylfluoride (PMSF) (Sigma-aldrich, P7626). Total protein was quantified via Bicinchoninic Acid Assay (Pierce), denatured with 2× laemmli buffer (Sigma-aldrich, 38733) and heated to 95°C for 5 min. 20 µg total protein was loaded onto each well of a 7.5% polyacrylamide gel (BioRad), separated via electrophoresis, and transferred to PVDF membrane (Sigma-aldrich, P2438). Blots were stained accordingly with anti-Rorγt (Ebioscience, 14-6981-82), anti-MPO (R&D systems), and anti-β-actin (Cell signaling, 4967), and then with HRP-conjugated secondary antibodies (Santa cruz biotechnologies). All blots were developed using the GE Healthcare ECL Western Blotting Detection Reagent (Amersham). The protein transfer zone was shown on photosensitive film (Hyperfilm, GE Healthcare) by rapidly developing and fixing in the darkroom, and the optical density was analyzed with AlphaView analysis software.