Novocastra peroxidase dab kit

Liver and kidney samples were fixed in a 4% formaldehyde solution in PBS, embedded in paraffin, and stained using routine hematoxylin and eosin stain (H&E). An Olympus BX43 microscope with a digital camera (Olympus XC30, Olympus, Hamburg, Germany) was used for the microscopy analyses of the slides. Prior to immunochemistry, the sections were deparaffinized and rehydrated. The primary antibody used was TNF-α (1:200 dilution), and then specific secondary antibodies were applied. The immunoreaction was detected using a Novocastra Peroxidase/DAB kit (Leica Biosystems, Nussloch, Germany). The negative control sections were stained with irrelevant immunoglobulins and analyzed under a bright-field microscope. After dehydration, tissue sections were mounted with BioMount mounting medium and analyzed by light microscopy using the Olympus System Microscope Model BX43 equipped with a digital camera.

Immunohistochemistry was done on 5 µm paraffin-embedded eye sections, previously deparafinized and rehydrated using a standard technique. Primary antibodies diluted 1:200: mouse monoclonal caspase-3 (sc-271759; Santa Cruz Biotechnology; Dallas, TX, USA), rabbit polyclonal NF-kB (sc-109; Santa Cruz Biotechnology; Dallas, TX, USA), mouse monoclonal TNF-α (ab 1793; Abcam PLC., Cambridge, UK) were incubated overnight la 4 °C.
Novocastra Peroxidase/DAB kit (Leica Biosystems, Nussloch, Germany) was used to detect immunoreactions, according to the manufacturer’s instructions. The substitution of primary antibodies with irrelevant immunoglobulins of matched isotype was used to stain negative control sections and all were analysed under bright-field microscopy.

Adipose tissue regeneration was assessed by histological and immunohistochemical analyzes (adipogenic markers).
For the histopathological study, explant samples were fixed in phosphate-buffered formaldehyde solution (4%, pH 7.2, 0.05 M). After dehydration and clarification, samples were embedded in paraffin. After sectioning at 5 µm thickness, sections were stained with hematoxylin and eosin (H&E) and Gomori’ s trichrome kit (Leica Biosystems, 38016SS1, Nussloch, Germany) according to the manufacturer’s protocol.
For the immunohistochemical studies the paraffin-embedded explant tissue sections were previously deparaffinized and rehydrated using a standard technique. After heat-mediated antigen retrieval in citrate buffer (pH 6.5), the sections were incubated overnight at 4 °C with the primary antibody. Rabbit polyclonal anti-tumor necrosis factor (TNF)-α diluted 1:100 (Santa Cruz, CA, USA) was used as a primary antibody. Immuno-reactions were visualized employing a Novocastra Peroxidase/DAB kit (Leica Biosystems, Nussloch, Germany) according to the manufacturers’ instructions.
All the microscopic sections were analyzed with an Olympus BX43 microscope equipped with a digital camera Olympus XC30 and CellSense software.