The xenograft mice were sacrificed, and the tumors were freshly collected to perform the experiment. Then, formalin-fixed and paraffin-embedded sections of 5 µm thickness were dried at 60 °C for 30 min, deparaffinized in xylene, rehydrated through graded alcohols, and immersed for 15 min in PBS buffer. For antigen retrieval, the sections were microwaved in 0.01 M citrate buffer (pH 6.0) for 20 min. After that, the endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase in methanol for 30 min. The sections were incubated for 60 min with 5% BSA and 0.3% Triton X-100 in PBS-T to block nonspecific staining and then incubated with indicated primary antibodies overnight. Alexa Fluor® 555 (Abcam, Cat#ab150074, UK), Alexa Fluor® 647 (Abcam, Cat#ab150115, UK), and Alexa Fluor® 647 (Abcam, Cat#ab150131, UK) were diluted in IF-blocking buffer. The specimens were washed with PBS, and then the nuclei were stained with DAPI (Invitrogen, Cat#P36931, USA) for 20 min. The slides were mounted with VECTASHIELD Antifade Mounting Medium for Fluorescence (Vector Laboratories, Cat#H-1000, Burlingame, CA, USA). The samples were imaged using a laser-scanning confocal microscope (Nikon A1), and fluorescence was analyzed. Three to four images were randomly taken per slide for analysis.
Vectashield antifade mounting medium for fluorescence
Manufactured by Vector Laboratories
Sourced in United States
Vectashield is an antifade mounting medium for fluorescence applications. It is designed to retard the fading of fluorescent signals, thereby preserving the integrity of the fluorescent signal during microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Alexa fluor 647, by Abcam (1 mentions)
Alexa fluor 555, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti cith3, by Abcam (1 mentions)
Zen black software, by Zeiss (1 mentions)
Confocal microscope lsm700, by Zeiss (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Mouse anti nc82, by Developmental Studies Hybridoma Bank (1 mentions)
3 protocols using vectashield antifade mounting medium for fluorescence
1
Immunofluorescent Staining of Xenograft Tumor Samples
2
Immunostaining of Frozen Tissue Sections
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Frozen tissue sections were thawed for 30 min at room temperature and washed with PBS before permeabilization with 0.25% Triton X-100 or 0.5% saponin in PBS for 10 min. After permeabilization, sections were washed with PBS again and blocked with 3% BSA at room temperature for 30 min. Tissue sections were stained with primary antibodies (anti-cit-H3 (Abcam), anti-DNA/H1 (Merck) and anti-GR-1 (produced in-house 59 )) diluted in blocking buffer (3% BSA in PBS) for 1 h 30 min in a humidified chamber, after which they were washed with PBS-Tween-20 (0.05%) and stained with secondary antibodies for 1 h at room temperature in the dark. Sections were washed as before and counterstained with 1 µg ml -1 DAPI and mounted in Vectashield antifade mounting medium for fluorescence (Vectorlabs). Coverslips were sealed with nail polish and slides were stored in the dark at 4 °C until imaging. Fluorescence was visualised using the Zeiss confocal LSM700 microscope and Zen Black software (Zeiss). The quantification of positive area (area + %) for each channel was conducted using Fiji software. Image preprocessing included utilizing the built-in 'Moments' algorithm for thresholding each channel.
3
Immunostaining of Whole-Mount Drosophila Brains
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The following procedure was used for immunostaining whole-mount brains. First, brains dissected from 3-7 day old of female flies were fixed with 4 % paraformaldehyde for 2 hours at room temperature and followed by extensive washes with washing buffer (0.3 % PBT) for 4 x 20 min. After washes, brains were incubated in blocking buffer (5 % normal goat serum) for 1.5 hours. Next, the brains were incubated in primary and secondary antibodies at 4 C for 48 hours each, with extensive washes (0.3 % PBT, 4 X 20 min) between steps. Primary antibodies used here included Rabbit anti-GFP (1:1000, Invitrogen) and mouse anti-nc82 (1:30, Developmental Studies Hybridoma Bank). Secondary antibodies used here were AF488 goat α-rabbit (1:400, Invitrogen), AF568 goat α-mouse (1:400, Invitrogen), and AF647 goat α-mouse (1:400, Invitrogen). The brains were then mounted on microscope slides with Vectashield antifade mounting medium for fluorescence (Vector Laboratories). Confocal laser scanning micrographs were obtained using a Zeiss LSM880 confocal microscope with a 20X (1.0 N.A.) water immersion objective. Spacing between individual planes was 1 μm and the pixel resolution was set to 1024 x 1024.
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