Anti cd68 antibody

To identify TREM-1-expressing inflammatory cells in lung tissues, we obtained lung specimens from two patients with pulmonary TB that were diagnosed based on pathologic characteristics and positive TB cultures of lung specimens. Both their lung specimens showed necrotizing granulomatous inflammation with the presence of mycobacterium, and the in-house TB polymerase chain reaction results were positive. IHC staining for TREM-1 expression analysis was carried out in the core facility of Taipei Veterans General Hospital. In short, 4-μm sections were prepared from formalin-fixed, paraffin-embedded tissues and the IHC staining was performed on a Leica Bond-MAX system (automatic IHC staining system). After deparaffinization, sections were pre-treated using heat-mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 30 minutes. The section was then incubated with a human anti-TREM-1 antibody (ab93717, Abcam Inc.) at 10 µg/mL (diluted 1/100) for 60 minutes at room temperature, and detected using an HRP-conjugated compact polymer system (Anti-Rabbit IgG–Poly-HRP). The section was blocked with peroxide block (Bond Polymer Refine Detection, Leica Biosystems) for 5 minutes. DAB was used as the chromogen. IHC staining with anti-CD68 antibody (Dako, Glostrup, Denmark) was also performed to identify the presence of human macrophages in lung specimens.

After sacrifice, mouse organs were fixed in 4% buffered formaldehyde for 24 h, rinsed with PBS, dehydrated in a series of graded ethanol and embedded in paraffin. Sections of 5 μm thickness were cut and stained with haematoxylin and eosin. For immunohistochemistry 5 μm thick sections were cut, dewaxed, microwaved in Target Retrieval Solution (DAKO) for 2 x 4 min and cooled down to room temperature for 40 min. After washing with Tris-buffered saline (TBS), non-specific binding was blocked by incubating sections in 10% normal swine serum (DAKO) for 30 min at room temperature. Slides were incubated with anti-CD68 antibody (ABCAM ab955) at a dilution of 1 μg/ml for 60 min (RT), followed by a biotinylated rabbit anti mouse antibody (DakoCytomation) at a dilution of 1:200 for 30 min. After careful washes in TBS, an incubation with an avidin-alkaline phosphatase complex (ABC kit, Vectastain, Vector) for 30 min followed and thereafter, additional washes in TBS were performed. Alkaline phosphatase activity was visualized using Liquid Permanent Red (LPR) Substrate-Chromogen (DAKO) for 15 min. After washing with water, slides were counterstained with Mayer's hemalum diluted 1:1 in water for ten seconds, blued under water and mounted with Eukitt^® (Sigma).

Paraffin embedded tissues were analyzed using immunohistochemical staining [49 (link)] with the following primary antibodies: anti-CD68 antibody (DAKO, Carpinteria, CA), anti-α-SMA antibody (DAKO, Carpinteria, CA), anti-CCL7 antibody (Gen Way Biotech, San Diego, CA), anti-COL3A1 antibody (Bioss, Beijing, China), anti-FOXP3 antibody (Biolegend, San Diego, CA) and rabbit anti-Mouse CD68 antibody (Bioss, Beijing, China). For quantification of tumor stromal cells within the tumor area, CD68 was used as a pan-macrophage marker, α-SMA was used to detect cancer activated fibroblasts adjacent to RCC cells [50 (link)], FOXP3 was used as a specific marker for regulatory T cells (Tregs) [22 (link)], and mast cells were assessed using the routine toluidine blue staining method [21 (link)]. Each tumor section was evaluated by using 20× objective lens, and five independent areas with the most abundant positive cells were selected, digitally photographed, and manually counted under a microscope. The average positive cell counts for each patient were used for statistical analysis. For quantification of CD68⁺ cells in CDX xenografts, four sections from each xenograft were randomly selected and quantified as described above, the average positive cells for each mouse were used for statistical analysis. Results were confirmed by two pathologists in a double-blind analysis.