Ab207229

4% Paraformaldehyde solution was used to fix fresh tissue samples, which were then embedded in paraffin. Paraffin sections were cut at a thickness of 2μm for Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining. Evaluation of NIP histological characteristics and determination of basement membrane thickness was performed via H&E staining. The distribution of MMPs and neutrophils was detected by IHC. Briefly, sections were incubated with 1X pH 6.0 antigen retrieval buffer (Abcam, USA) and 3% hydrogen peroxide (Sigma Aldrich, USA) for eliminating endogenous peroxidase. Sections were then blocked in 10% goat serum (Abcam, USA) and incubated overnight in the dark at 4°C with primary antibodies of MMP-1 (1:200, Abcam, ab137332), MMP-7 (1:200, Abcam, ab207229), MMP-9 (1:200, Abcam, ab76003), TIMP-1 (1:200, Abcam, ab211962), TIMP-3 (1:200, Abcam, ab39184), neutrophil elastase (1:1000 clone NP57 Dako, Glostrup, Denmark), HIF-1α (1:200, Abcam, ab179483). Next, all sections were incubated with the Dako EnVision1 System-HRP (Dako) at room temperature for 30 minutes. For color development, Diaminobenzidine was used as a substrate, and all sections were counterstained by hematoxylin.

Protein was extracted from fresh NIP tissue using ice-cold RIPA lysis buffer (Abcam) mixture containing protease and phosphatase inhibitor (Sigma Aldrich). After 30 minutes of incubation on ice with shaking, followed by centrifugation at 14,000 rpm, the supernatant containing the protein was aspirated for use in Western blot analysis. Protein concentration was first determined using BCA protein assay (Thermo Fisher, USA); then, 4X laemmli sample buffer was added to the protein samples according to the manufacturer’s instructions, and heated at 95°C for 10 minutes. 30 μg total protein from each sample was subjected to electrophoresis in 10% SDS-PAGE gel and then transferred onto a 0.22μm PVDF membrane (Millipore, USA). After 2 hours non-specific blocking with 5% skimmed milk, primary antibody of MMP-1 (1:2000, Abcam, ab137332), MMP-7 (1:2000, Abcam, ab207229), MMP-9 (1:4000, Abcam, ab76003), TIMP-1 (1:2000, Abcam. Ab109125), TIMP-3 (1:2000, Abcam, ab39184), GAPDH (1:5000, Abcam, ab186930) were added to the membrane and incubated at 4°C overnight. HRP-conjugated anti-rabbit IgG or anti-mouse IgG were then added as secondary antibody at room temperature for 1 hour. After secondary antibody staining, ECL reagent (Bio-Rad, USA) was used to visualize the blots. Densitometry was performed with ImageJ software to quantify the protein amounts of each sample.

Nuclear and cytoplasmic fractionation of BMDM or muscle tissue lysates was carried out according to manufacturer instructions (Abcam, ab113474). Briefly, cells were collected with cell scraper and pelleted by centrifugation. Pellets were re-suspended in the pre-extraction buffer. After a centrifugation, supernatant containing cytoplasmic fraction was frozen down and stored. The pellet was again centrifuged and re-suspended in nuclear extraction buffer to generate the nuclear fraction. The nuclear fraction was used for HIF-1α, STAT3, and NF-κB p65 transcription assays according to the manufacturer’s instructions (Abcam; ab133104, ab207229, and ab133112, respectively).