Anti α sma a2547

Left lung tissue samples were evaluated for immunohistochemical localization of F4/80 and α-SMA (α-smooth muscle actin). The antibodies were obtained from the following sources: anti-mouse F4/80 (MCA497G) from AbD Serotec (Kidlington, UK) and anti-α-SMA (A2547) from Sigma-Aldrich (St. Louis, USA). Secondary antibodies conjugated with horseradish peroxidase (HRP) were all obtained from R&D Systems (Minneapolis, USA). To determine the specificity of staining, no primary antibody was used followed by incubation of section with secondary antibodies and detection reagents (18 (link)).

Paraffin-embedded liver sections were deparaffinized with xylene and dehydrated in gradually decreasing concentrations of ethanol. Immunohistochemical staining was performed according to the described procedure [6 (link),38 (link)]. For immunohistochemical staining, the liver sections were incubated with a primary antibody (1:100 dilution) for 1 h at 37 °C. Primary antibodies used were as follows: antifibronection (BD Biosciences, San Jose, CA, USA) and anti-α-SMA (A2547, Sigma-Aldrich, St. Louis, MO, USA). After 3 serial washes with PBS, the sections were processed by an indirect immunoperoxidase technique using a commercial Envision System kit (DAKO, Carpinteria, CA, USA). Immunohistochemical images were viewed with an Eclipse 80i microscope (Nikon, Tokyo, Japan).

Frozen sections of the aortic root were fixed in ice-cold acetone for 10 min, dried under a ventilator, and washed with PBS. Sections were blocked in 4% FCS with Avidin D solution (Avidin/Biotin Blocking Kit; Vector Laboratories) for 30 min. Primary antibodies were anti-mouse macrophages/monocytes (MCA519GT, Serotec), anti- active caspase 3 (AF835, R&D Systems), anti- α-SMA (A2547, Sigma-Aldrich), and anti- KLF4 (NBP2-24749, Novus Biological). Biotinylated secondary antibodies, ABC Kit Vectastain Elite, and DAB substrate (PerkinElmer, Vector, and Dako) were used. After counterstaining with haematoxylin sections were mounted with Entellon (MERCK) mounting medium. Stained area was measured using Adobe Photoshop or QuPath^{44 (link)}.

Three-micrometre-thick sections of the paraffin-embedded tumour samples were transferred to gelatinized micro-slides and air-dried overnight at 37 °C. They were deparaffinized in xylene (three changes of medium) and rehydrated in ethanol (100% through 30%). They were then washed three times in distilled water and twice in Tris-buffered saline (TBS) [50 mmol/L Tris/HCL (pH 7.4), containing 100 mmol/L sodium chloride]. To block non-specific binding, the sections were incubated in TBS containing 1% casein (SIGMA 8654) for 10 min. They were then exposed to primary antibodies against MMP-2, MMP-7, MMP-9, MMP-13, α-smooth-muscle actin (anti-α-SMA) (A-2547, Sigma-Aldrich-Chemie, Gmbh), diluted 1:200 in TBS, and rabbit anti-fibronectin (F 3648 Sigma-Aldrich-Chemie, Gmbh), diluted 1:200 in TBS. The latter two antibodies were applied to identify stromal-muscle cells and fibroblasts, respectively.

Five µm sections were deparaffinized, treated with 0.3% H₂O₂ to block endogenous peroxidases, and rehydrated. Depending on the protein targeted, sections were treated 20 min in 0.01 M sodium citrate pH6 at 95 °C for heat-induced antigen retrieval, before being blocked with pre-immune serum and incubated with primary antibodies for 1–4 hours. Secondary antibody system was composed of biotinylated secondary antibody, Vectastain ABC HRP kit (PK-4000) and Vector SG HRP substrate kit (SK-4700) (Vector Laboratories, Burlingame, CA, USA). Counter-staining was done with Nuclear Fast Red (#6070-5 G) (Fluka, Buchs, Switzerland). Primary antibodies were: anti-α-SMA (A2547) (Sigma-Aldrich, Darmstadt, Germany), anti-tenascin C (LS-C313183) (LifeSpan BioSciences Inc., Seattle, WA, USA). For α-SMA antibody, the Mouse on Mouse Detection Kit was used (BMK-2202) (Vector Laboratories, Burlingame, CA, USA). Pictures of three different experiments were evaluated by three different examiners (SG, TPC and JCS) for each immunohistochemical staining. Criteria for the evaluation of differences between genotypes were to be able to sort the pictures by genotype, not knowing to which animal it belonged.