Evans blue dye (solution at 5 mg/ml, Sigma‐Aldrich) was injected 24 h intraperitoneally before sacrifice (10 μl per g of mouse). Positivity for Evans blue dye was revealed through its fluorescence after excitation at 546 nm. Sections were fixed with acetone (VWR) for 10 min at −20 °C, permeabilized in 0.5% Triton X‐100 (Sigma‐Aldrich) for 10 min, blocked for 1 h in 4% BSA, and incubated overnight with rabbit anti‐laminin antibody (1:300, Sigma‐Aldrich) to reveal myofiber outlines. The day after, sections were washed, incubated with a goat anti‐rabbit 488 secondary antibody (1:250, Jackson ImmunoResearch) and Hoechst (1:500, Sigma‐Aldrich) in PBS for 45 min at room temperature, then washed 4 times for 5 min with PBS and mounted with Fluorescence Mounting Medium (Sigma‐Aldrich). Measurement of the percentage of Evans blue dye uptake was performed by counting the number of Evans blue dye‐positive fibers on total muscle section reconstructions, using ImageJ software (NIH, Bethesda, MD, USA).
Fluorescence mounting medium
Manufactured by Merck Group
Sourced in United Kingdom, United States, Italy
Fluorescence mounting medium is a clear, viscous solution used to protect fluorescently labeled samples and maintain their fluorescence during microscopic examination. It is designed to be applied between a slide and a coverslip to create a stable, refractive index-matched environment for optimal fluorescence imaging.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp8 confocal microscope, by Leica (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Acetone, by Avantor (1 mentions)
Hoechst, by Merck Group (1 mentions)
Rabbit anti laminin antibody, by Merck Group (1 mentions)
Evans blue dye, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Neutral resin, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ihc wash buffer, by Beyotime (1 mentions)
Lsm 510 laser scanning microscope, by Zeiss (1 mentions)
Live dead viability cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Connexin 43, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Tunel brightred apoptosis detection kit, by Vazyme (1 mentions)
8 protocols using fluorescence mounting medium
1
Assessing Vascular Permeability in Mice
2
Immunofluorescence Analysis of Bone Markers
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The paraffin embedded calvarial sections were deparaffinized and rehydrated in alcohol gradient (100%, 96% and 70% volume). Sections were washed and antigen unmasking was performed with sodium citrate buffer (pH 6.0). Slides were blocked with 1% bovine serum albumin (BSA) and 5% normal goat serum in phosphate buffered saline (PBS) solution and incubated for 1 h at room temperature, followed by three washes with PBS. Primary antibodies BMP, Runx-2, OPN and OCN were added in 1% BSA solution and incubated overnight at 4 °C at 1:100 dilution. The slides were washed and then incubated with the corresponding secondary antibodies diluted 1:500 (Alexa Fluor dye conjugated) in the appropriate blocking solution for 1 h at room temperature in dark. Counterstaining of nuclei was preformed with DAPI. Stained slides were mounted in fluorescence mounting medium (Sigma Chemical) and analyzed under a Leica TCS SP8 confocal microscope.
3
Immunolocalization of Fasciola gigantica ES Antigens
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Liver tissue infected with Fasciola gigantica were collected from the naturally infected buffalo slaughtered at local abattoir. Also livers from experimentally infected male New Zealand white rabbits, sacrificed by intravenous injection of 100 mg/kg (body weight) sodium pentobarbital, were obtained and fixed in 10% phosphate buffered formalin solution. About 5 μm thick sections of liver tissue infected with parasite were cut using rotary microtome. The sections were deparaffinised in xylene and rehydrated in descending grades of ethanol. The sections were washed thrice with cold 100mM Tris Buffered saline pH 7.4 and incubated in 0.05% (w/v) protease type XXIV, prepared in TBS, for 10 minutes at 37°C for antigen retrieval. Following washing thrice with TBS blocking was done with 50% FBS in TBS for 2 h at RT. The sections were washed again and incubated in 1:500 diluted rabbit anti-ES primary antibody for 16–18 h at 4°C. The sections were washed thrice and incubated in 1:10000 diluted FITC conjugated anti-rabbit IgG secondary antibody for 6 h at RT. The sections were washed thrice and counterstained using 1:10000 diluted phalloidin TRITC secondary antibody for 6 h at RT. Finally sections were mounted in Fluorescence mounting medium containing anti fade (Sigma). The slides were observed at USIF, AMU, Aligarh on a Zeiss Confocal microscope (LSM 780) and digital images were saved.
4
Immunohistochemistry Protocol for Tissue Sections
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Briefly, fresh tissues were fixed via immersion in 4% paraformaldehyde (Sigma-Aldrich) at RT for 30 min. The tissues were then subjected to gradient ethanol dehydration, embedded in paraffin, sectioned (thickness, 6 μm), and dewaxed in xylene. Next, the tissue sections were blocked at 37°C for 30 min in immunohistochemistry (IHC) blocking solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China). After removal of the blocking solution, the tissue sections were washed 3 times at RT for 5 min each using IHC wash buffer (Beyotime Biotechnology Co., Ltd.). Subsequently, the tissue sections were incubated with primary antibodies (Table II ) at 37°C for 45 min. After removal of the primary antibodies, the tissue sections were washed 3 times at RT for 5 min each using IHC wash buffer (Beyotime Biotechnology Co., Ltd.). The tissue sections were then incubated with secondary antibodies (Table I ) at 37°C for 45 min. The secondary antibodies were subsequently discarded, and the tissue sections were washed 3 additional times at RT for 5 min each using IHC wash buffer (Beyotime Biotechnology Co., Ltd.). Finally, the tissue sections were mounted using neutral resin (Sigma-Aldrich) or fluorescence mounting medium (Sigma-Aldrich).
5
Cellular Uptake of Fluorescent Cisplatin
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HepG2 and HepG2/cr cells were grown overnight on 4-well glass Lab-Tek chamber slides (Nalgen Nunc International, Rochester, NY, USA). They were then incubated with Alexa Fluor 546-labelled cisplatin (F-cisplatin; Molecular probes, Life Technologies, UK) at a final concentration of 200 U/mL (1 unit is defined as the reagent solution required to label 25 ng of DNA in vitro at 37 °C, for up to 48 h). The cells were then washed again with ice-cold PBS and fixed with ice-cold 70% ethanol for 15 min at −20 °C, before being washed again with ice-cold PBS. Cover slips were then mounted atop, using fluorescence mounting medium (Sigma Aldrich, UK), before being analysed at ×400 magnification, using an LSM510 laser-scanning microscope (Zeiss, Heidelberg, Germany).
6
Osteoblast Viability Evaluation
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Osteoblast viability after 24 h of incubation was explored among the experimental substrates using a live/dead viability/cytotoxicity assay kit (Gibco-Invitrogen, Carlsbad, CA, USA), mixing 1mM calcein-AM and 2 mg/mL of ethidium homodimer-1 following the manufacturer’s instructions. Afterwards, the materials were inverted and mounted onto cover slides with a fluorescence mounting medium (Sigma-Aldrich, USA), characterized and photographed with a green (live) and red (dead) filter under an inverted fluorescence microscope (ZOE, Bio-Rad, Hercules, CA, USA). At least five fields were randomly imaged.
7
Quantifying Retinal Connexin 43 Expression
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The paraffin embedded retina sections were deparaffinized and rehydrated in an alcohol gradient (100%, 96% and 70% volume). The sections were washed, and antigen unmasking was performed with sodium citrate buffer (pH 6.0). Slides were blocked with 1% bovine serum albumin (BSA) and 5% normal goat serum in phosphate buffered saline (PBS) solution, washed with PBS and incubated with primary antibody, connexin 43 (sc-59949 Santa Cruz, US), for 2 h at 1:200 dilution. The slides were washed and then incubated with the corresponding secondary antibodies diluted 1:500 (Alexa Fluor dye conjugated) in the appropriate blocking solution for 1 h at room temperature in the dark. Counterstaining of nuclei was performed with DAPI. Stained slides were mounted in fluorescence mounting medium (Sigma Chemical, Italy) and analyzed under a Leica TCS SP8 confocal microscope. The intensity of the connexin 43 fluorescence was analyzed with ImageJ64 software (NIH, Bethesda, Maryland, USA). Five fields were selected randomly from each retina section. These values are presented as percentage fluorescence compared to the control group, which is set at 100%.
8
TUNEL Assay for Apoptosis Detection
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The TUNEL assay was performed by TUNEL BrightRed Apoptosis Detection Kit (A113-01; Vazyme). Briefly, sections were permeabilized by protein K and labeled with rTdT reaction mix for 1 h at 37°C. The reaction was stopped by 1× PBS. After washing with PBS, the sections were incubated in 2 µg/ml of DAPI (D1306; Molecular Probes) for 10 min. Sections were mounted on slices with Fluorescence Mounting Medium (F4680; Sigma-Aldrich). Images were obtained using a laser scanning confocal microscope LSM780 (Carl Zeiss).
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