Mice were euthanized by cervical dislocation and the eyes enucleated and fixed in 4% of Roti-Histofix (Carl Roth, Karlsruhe, Germany) for 3 h at room temperature (RT). The dissected RPE/choroidal flat mounts were permeabilized and blocked overnight in Perm/Block buffer (5% normal donkey serum (NDS), 0.2% bovine serum albumin (BSA), 0.3% Triton X-100 in PBS) at 4 °C. RPE/choroidal flat mounts were stained in addition with FITC-conjugated isolectin B4 from Bandeiraea simplicifolia (BS, 1:100 diluted in Perm/Block, L2895, Sigma-Aldrich). After several washing steps, retinal and RPE/choroidal flat mounts were mounted on a microscope slide and embedded with fluorescence mounting medium (Vectashield HardSet H-1400, Vector Labs, Newark, California, USA). Images were taken with a Zeiss Imager. M2 equipped with an ApoTome.2 (Carl Zeiss, Oberkochen, Germany). Areas of CNV in RPE/choroidal flat mounts were measured with the spline function of the graphic tool included in the ZEN software (Zeiss). The average CNV area per eye was calculated.
Vectashield hardset h 1400
Manufactured by Vector Laboratories
Sourced in United States
Vectashield HardSet H-1400 is a mounting medium designed for use with fluorescence-based microscopy techniques. It is formulated to provide a clear, hardened mounting surface for fluorescently-labeled samples.
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Lab products found in correlation
2 protocols using vectashield hardset h 1400
1
Quantification of Choroidal Neovascularization in Mice
2
Quantifying Podocyte Density via WT-1 Immunofluorescence
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Podocytes were stained by immunofluorescnce using WT‐1 as a marker. Slices, 4 μm thick, were de‐paraffinized, blocked in goat serum (G9023; Sigma, Rehovot, Israel) and incubated with anti‐WT‐1 Ab conjugated to Alexafluor 647 (sc393498 AF647; Santa Cruz, Dallas, TX, USA 1:500), diluted in PBS with 1% BSA (A2153; Sigma) and left overnight at 4°C. Slides were washed twice in PBS, mounted using VECTASHIELD® HardSet (H‐1400; Vector Labs, Burlingame, CA, USA) and kept at 4°C. Stained sections were examined and photographed by a fluorescence microscope (Eclipse Ni‐U, NiKOn, Tokyo, Japan). The average number of podocytes in three different glomeruli was calculated for each animal.
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