Anti f4 80 and anti cd206 antibodies

To determine the newly formed vessels in gastrocnemius muscles after the hADSCs administration, the frozen sections were stained immunohistochemically. The frozen sections were incubated with antibodies directed against rat CD31 and rabbit CD146, α-SMA (Abcam). Additionally anti-F4/80 and anti-CD206 antibodies (Abcam) were used to identify M2 macrophages. Subsequently, sections were incubated with a secondary antibodies conjugated with Texas Red, FITC, or Alexa Fluor (Abcam). Sections were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Imaging of the fluorescence of the stained sections was performed with the confocal microscope LSM710.

After 3, 7 and 14 days after surgery, mice were sacrificed and gastrocnemius muscles were dissected, frozen in liquid nitrogen and sectioned at 5 μm thickness. The capillaries were visualized by immunofluorescent staining with anti‐CD31 (Abcam). Anti‐F4/80 and anti‐CD206 antibodies (Abcam) were used to identify M2 macrophages. Respective secondary antibody conjugated with FITC or Texas Red (Vector Laboratories) was used. The slides were coverslipped using VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Imaging of the fluorescence of the stained sections was performed with the confocal microscope LSM710. Frozen sections were examined histochemically (haematoxylin/eosin staining; Sigma‐Aldrich). Analyses of the specimens were conducted using Nikon Eclipse 80i microscope (Nikon Instruments Inc).

The tumors were collected, frozen in liquid nitrogen, and sectioned into 5 μm slices. Macrophages in the tumor sections were stained with anti-F4/80 and anti-CD206 antibodies (Abcam, Cambridge, UK), followed by appropriate fluorochrome-conjugated (Alexa Fluor 594 (Abcam) and FITC (Vector Laboratories, Burlingame, CA, USA) secondary antibodies. The cytotoxic T lymphocytes were stained with anti-CD8α (Abcam) and subsequently with Alexa Fluor 594-conjugated secondary antibodies (Abcam). For the immunohistochemical analyses of pericytes coverage, tumor blood vessels sections were incubated with anti-α-Smooth Muscle Actin and anti-CD31 antibodies (Abcam) and subsequently with Texas Red and FITC-conjugated secondary antibodies (Vector Laboratories) [13 (link)]. All the sections were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Microscopic observations were performed using a LSM710 confocal microscope (Carl Zeiss Microscopy GmbH). The obtained confocal images were analyzed with ImageJ 1.48v (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, LOCI, University of Wisconsin, USA) and the results were expressed as the percentage of area [%].