**2.8.3 Immunohistochemistry on paraffin sections**

While whole mount immunohistochemistry is a valuable tool to study vascular structure and functionality, different methods are advantageous to draw quantitative conclusions regarding the number of blood vessels per adipocyte. For this purpose, the use of thin sections of paraffin-embedded adipose tissue is recommended. In this approach, in contrast to whole mount immunohistochemistry where the thickness of the tissue varies as it is cut by hand, rather uniform tissue sections are prepared using a microtome. Immunohistochemistry on paraffin sections is based on the same principle as whole mount immunohistochemistry: the tissue is incubated with a primary antibody against a protein of interest. A fluorochrome or horse radish peroxidase (HRP)-labeled secondary antibody is then used to visualize the protein (Fig. 7 and 8).

Fig. 7. Schematic representation of immunohistochemistry on paraffin sections using fluorescence-based detection of signals.

It is helpful to counterstain the tissue with DAPI, propidium iodide or Hoechst (for fluorescent stainings) or hematoxylin (for chromogenic HRP stainings) to visualize the nuclei of cells and thereby provide additional information on the structure of the tissue. To study active angiogenesis, proliferating endothelial cells can be visualized by double staining with antibodies against an endothelial cell marker, such as CD31, and a

proliferation marker, such as PCNA or Ki67. Another option is injection of 5-bromo-2'-

While whole mount immunohistochemistry is a valuable tool to study vascular structure and functionality, different methods are advantageous to draw quantitative conclusions regarding the number of blood vessels per adipocyte. For this purpose, the use of thin sections of paraffin-embedded adipose tissue is recommended. In this approach, in contrast to whole mount immunohistochemistry where the thickness of the tissue varies as it is cut by hand, rather uniform tissue sections are prepared using a microtome. Immunohistochemistry on paraffin sections is based on the same principle as whole mount immunohistochemistry: the tissue is incubated with a primary antibody against a protein of interest. A fluorochrome or horse radish peroxidase (HRP)-labeled secondary antibody is then used to visualize the

Fig. 7. Schematic representation of immunohistochemistry on paraffin sections using

It is helpful to counterstain the tissue with DAPI, propidium iodide or Hoechst (for fluorescent stainings) or hematoxylin (for chromogenic HRP stainings) to visualize the nuclei of cells and thereby provide additional information on the structure of the tissue. To study active angiogenesis, proliferating endothelial cells can be visualized by double staining with antibodies against an endothelial cell marker, such as CD31, and a proliferation marker, such as PCNA or Ki67. Another option is injection of 5-bromo-2'-

fluorescence-based detection of signals.

**2.8.3 Immunohistochemistry on paraffin sections** 

protein (Fig. 7 and 8).

deoxyuridine (BrdU) into the tail vain of a mouse 1 min prior to sacrificing the animal. This synthetic thymidine analogue is incorporated into the DNA of proliferating cells. Using an anti-BrdU antibody thereby allows for detection of actively replicating cells.

Fig. 8. Schematic representation of immunohistochemistry on paraffin sections using chromogenic detection of signals. The organic compound 3,3'-Diaminobenzidine is oxidized by HRP resulting in a brown color.

### **2.8.4 Immunohistochemisty on cryosections**

Some antigens are masked by paraffin fixation, and in these cases staining has to be done on non-fixed, frozen tissues instead. Dissected adipose or other tissues should be embedded immediately in a plastic cryomold, snap frozen on dry ice and stored at -80°C until staining. Sectioning of cryo-embedded adipose tissue is slightly more challenging than other tissues due to its rather soft integrity. Hence, is it critical to lower the temperature of the cryotome to -30°C before sectioning cryo-embedded adipose tissue samples. Thin sections of 15 µm should be adhered on Superfrost Plus microscope slides, subsequently fixed with cold acetone and stained with specific primary antibodies. However, it is important to note, when staining for lymphatic vessels in the adipose tissue that LYVE-1 is not as widespread on the lymphatic endothelium as podoplanin in this particular tissue (Fig. 9). Here, unlike other tissues, LYVE-1 staining mainly detects inflammatory cells.

Fig. 9. Double cryosection immunostaining of the WAT of a mouse with podoplanin (red) and LYVE-1 (green). Podoplanin-positive lymphatic staining is indicated with arrowheads and LYVE-1-positive non- lymphatic staining is indicated with arrows. Scale bar = 100 μm.

### **2.8.5 Hypoxia staining**

Since hypoxia is one of the driving forces of angiogenesis, detection of the presence of hypoxia in tissues could also be performed. Hypoxyprobe-1 (pimonidazole hydrochloride) staining can be used to detect cell and tissue hypoxia. Pimonidazole hydrochloride has a molecular weight of 290.8 kD, ultraviolet absorbance at 324 nm and a plasma half-life of approximately 25 minutes in mice. The detection of hypoxia in tissues can be performed by intravenous or intraperitoneal injection or oral ingestion of pimonidazole hydrochloride at a dosage of 60 mg/kg, 15-90 minutes before sacrificing mice. Dissected tissues should be fixed in 4% PFA followed by paraffin embedding. Paraffin-embedded tissue sections of 3-5 µm is further stained using a peroxidase conjugated anti-pimonidazole antibodies and counterstained with nuclei staining.
