Mouse anti human cd20

Pathological assessment of stromal lymphocytic infiltration was carried out as described [18 (link)].
Infiltration of total leukocytes, T, B, CD8+ T cells and NK cells was analyzed immunohistochemically on FFPE tumor sections using the Automated Immunostainer. The following antibodies were used: mouse anti-human CD45 (1:200, Dako), rabbit anti-human CD3 (1:400, Dako), mouse anti-human CD20 (1:400, Dako), mouse anti-human CD8 (1:200, Dako), and mouse anti-human CD56 (1:400, NeoMarkers, Fremont, CA), respectively. Antigen retrieval was carried out using the Target Retrieval Solution pH9 (Dako). Immunoreactions were visualized using streptavidin-biotin-peroxidase, followed by counterstaining with Carazzi hematoxylin. Stained slides were digitized by a slide scanner (ImageScope XT, Aperio), and the virtual slides were subsequently evaluated using the ‘positive pixel count’ algorithm of Aperio ImageScope. The percentage of positive stromal cells was calculated as the number of positive pixels/μm². Data were divided into two groups (positive and negative) using median value as cut-off.

Cisplatin and Paclitaxel were obtained commercially from Sigma-Aldrich (St Louis, MO), PLX4720 and PD0325901 were purchased from Selleckchem (Houston, TX). They were dissolved in H₂O or DMSO and were stored at −20°C as 10 mM stocks.
Mouse anti-human IGF-1 neutralizing monoclonal antibody (clone Sm1.2 (IgG1))^{42 (link)–44 (link)} was obtained from Lifespan Biosciences (LS-C7504; Seattle, WA); goat anti-human FGF-2 neutralizing antibody was obtained from R&D Systems (AF-233-NA; Minneapolis, MN); rabbit anti-human FGFR-3 (ab10651), phospho-FGFR-3 (ab155960) and IGF-1 (ab9572) antibodies were obtained from Abcam Inc. (Cambridge, MA); mouse anti-human β-actin antibody was obtained from Sigma-Aldrich (A5316). Mouse anti-human CD20 was obtained from Dako (M0755; Carpinteria, CA). Mouse anti-human PAX5 (B-cell transcription factor; IgG1) was obtained from Leica Biosystems (PA0552; Buffalo Grove, IL). (Fluorochrome-conjugated antibodies for CD20 (11-0209), CD45 (12-9459), and CD146 (12-1469) were obtained from ebiosciences (San Diego, CA), CD133 (130-080-801) and CD271 (130-098-112) were obtained from Miltenyi Biotech (Auburn, CA) and FGFR-3 was obtained from R&D Systems (MAB766). CD271-dsRed promoter construct was obtained from Dr. Shi-Hua Li, Emory University, Atlanta, GA.

After deparaffinization and blocking steps, sections were incubated with rabbit anti-human CD31 (1:100, ab28364, Abcam), mouse anti-human CD20 (1:50, Dako), rabbit anti-human CD20 (1:400 Thermo Fisher), mouse anti-human VEGF (1:80, MA-13182, Thermo Fisher), and a species-matched isotype control. Slides were then washed and secondary staining was performed with donkey anti-mouse-Alexa-555 or goat anti-rabbit-Alexa-488 in the case of the double stain for CD20/CD31 and EBI-3. For double staining of CD20/VEGF, goat anti-mouse-Alexa-555 or goat anti-rabbit-Alexa-488 was used. All slides were counterstained with DAPI and examined under Carl Zeiss LSM 700.