To investigate the distribution of intravenously injected miR-PEI-NPs into the kidneys, liver, and spleen, Cy3-labeled double-strand oligos (Cy3-miR; Takara Bio Inc) were used to make a complex with PEI-NPs. These Cy3-miR-PEI-NPs were injected via the tail vein of mice with UUO-induced renal fibrosis. The kidneys, liver, and spleen were removed from each mouse 1 hour later. The tissue samples were embedded in Tissue-Tek® OCT (optimal cutting temperature) Compound and frozen in liquid nitrogen. Cryostat sections (5 μm thick) were mounted onto silane-coated glass slides (Matsunami Glass Ind Ltd, Osaka, Japan). Kidney tissues were incubated for 2 hours at room temperature with fluorescein-labeled Lotus tetragonolobus lectin (a proximal tubule marker) (Vector Laboratories Inc, Burlingame, CA, USA). Fluorescence was monitored by fluorescence microscopy (BH2-RFL-T3 and BX50; Olympus, Tokyo, Japan) and processed with Adobe Photoshop CS3 software (Adobe Systems Incorporated, San Jose, CA, USA).
Bh2 rfl t3
Manufactured by Olympus
Sourced in Japan
The BH2-RFL-T3 is a laboratory microscope from Olympus. It is designed for brightfield and reflected light microscopy applications. The core function of this product is to provide a platform for visual observation and analysis of samples under various illumination conditions.
Automatically generated - may contain errors
Lab products found in correlation
Acridine orange, by Thermo Fisher Scientific (3 mentions)
Fluorescein labeled lotus tetragonolobus lectin, by Vector Laboratories (2 mentions)
Silane coated glass slides, by Matsunami (2 mentions)
Bx51wi, by Olympus (2 mentions)
Uplan fln 40, by Olympus (2 mentions)
U mgfphq, by Olympus (2 mentions)
Cy5 4040c, by IDEX Corporation (2 mentions)
Rabbit anti tubulin β3, by BioLegend (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Methanol, by Fujifilm (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Ix51 inverted microscope, by Olympus (1 mentions)
Dcfh da, by Merck Group (1 mentions)
11 protocols using bh2 rfl t3
1
Visualization of Microrna-Nanoparticle Biodistribution
2
Characterization of Stem Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% paraformaldehyde for 20 minutes and incubated in 0.5% Triton X-100 for 10 minutes and 5% goat serum for 30 minutes. The primary antibodies included mouse anti-ABCG2 (1:100; Abcam Technologies, Cambridge, UK), rabbit anti-p63 (1:100; Abcam Technologies, Cambridge, UK), rabbit anti-CK14 (1:100; Abcam Technologies, Cambridge, UK), mouse anti-CK3 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Tubulin β3 (1:100; BioLegend, San Diego, CA, USA), mouse anti-CK1/10 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-MITF (1:50; Sigma-Aldrich Corporation, St. Louis, MO). Cells were incubated with the primary antibodies overnight at 4°C. Secondary antibodies (1:100; all obtained from Beijing Zhongshan Technologies, Beijing, China) coupled to fluorescein isothiocyanate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC) were subsequently applied for detection. The cells were then stained with 4, 6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Fluorescence was observed using a fluorescence microscope (model BH2RFL-T3, Olympus Corporation, Tokyo, Japan).
3
Immunofluorescence Staining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with methanol (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) in phosphate buffered saline solution (PBS) for 10 minutes. After washing in PBS, the cells were incubated for 30 minutes with 3% bovine serum albumin (BSA) in PBS containing 0.3% Triton × 20 (BSA/PBST with Tween 20 (PBST)) to block nonspecific staining. Then the cells were incubated for two hours at room temperature with specific primary Abs diluted in BSA/PBST. After further washing in PBS, the cells were incubated for one hour at room temperature with the appropriate secondary Abs diluted in BSA/PBST. Nuclei were counterstained with Hoechst 33342 (1:2000; Thermo Fisher Scientific) or DAPI (5 µM, Abcam). After further washing in PBS, examination was done under a fluorescence microscope (model BH2-RFL-T3 and BX50; Olympus, Tokyo, Japan).
4
Tracking Kidney siRNA Distribution
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the distribution of siRNAs that were systemically delivered by the tail vein injection approach in the kidney, Cy-3-labeled siRNAs (3 nmol) (Takara Bio Inc.) were systemically injected via the tail vein into renal fibrosis mice. One hour after injection, the kidneys were removed, cut and fixed at room temperature with 3% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, containing 25 mM CaCl2. Fixed tissues were immersed and embedded in Tissue-Tek optimal cutting temperature (OCT) compound, and frozen in liquid nitrogen. Cryostat sections 7 to 8 μm thick were mounted on silane-coated glass slides (Matsunami, Osaka, Japan) and air-dried. Then, the tissues were incubated for 2 hours at room temperature with fluorescein-labeled lotus tetragonolobus lectin (a proximal tubule marker) (Vector Labs, Burlingame, CA). Finally, fluorescence was detected by observation under a fluorescence microscope (models BH2-RFL-T3 and BX50, Olympus, Tokyo, Japan) and processed with Adobe Photoshop software (Adobe Systems, San Jose, CA).
5
Widefield Epifluorescence Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Widefield epifluorescent images were obtained on an Olympus IX51 inverted microscope with a mercury bulb (Olympus, BH2-RFL-T3) and white light source (Olympus, JH40100). Fluorescent images were obtained with GFP, Texas Red, and DAPI filter sets. For direct comparisons within a group, acquisition parameters of each channel were adjusted equally for all images.
Image processing was performed with the Fiji software package [33 –35 (link)] to combine color channels. For direct comparisons within a group, digital gain of each channel was adjusted equally for all images.
Image processing was performed with the Fiji software package [33 –35 (link)] to combine color channels. For direct comparisons within a group, digital gain of each channel was adjusted equally for all images.
6
Quantifying Intracellular ROS in Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular reactive oxygen species were measured, based on the reaction of oxidation of 2′,7′-dichlorofluoresce diacetate (DCFH-DA) to a fluorescent 2′,7′-dichlorofluorescein (DCF) by reactive oxygen species in the substrates. The cultured neurons on the coverslips, from 6–8 animals per group were treated with DCFH-DA (10 μmol/l, Sigma-Aldrich, USA) for 30 min at 37 °C in the dark, as reported17 (link). Then the cells were visualized and photographed using a microscope (BX51, Olympus) equipped with epifluorescence optics and a 100-W mercury lamp (Olympus BH2-RFLT3). The images were analyzed by ImageJ, a NIH software. The amount of reactive oxygen species was expressed as fluorescence intensity (FI). For each group in each of 6 individual tests, 50 individual neurons from 6–8 animals were analyzed.
7
Multicolor Fluorescence Imaging with Plasmonic Enhancement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were observed with an upright microscope (BX51WI; Olympus, Tokyo, Japan) using a 40× objective (UPLAN FLN ×40; Olympus, Tokyo, Japan). The light source was from an Hg lamp (BH2-RFL-T3; Olympus, Tokyo, Japan) and the detection camera was an electron multiplying charge-coupled device camera (EM-CCD, iXon; Andor, Belfast, UK). GFP (UMGFPHQ; Olympus, Tokyo, Japan) and Cy5 (Cy5-4040C; Semrock, New York, NY, USA) filter units were used for multicolor fluorescence imaging. 488-EGFR and APC-EpCAM were excited with GFP (460–480 nm) and Cy5 (605–645 nm) filters and detected with GFP (490–545 nm) and Cy5 (670–715 nm) filters, respectively. The plasmonic enhancement effect was seen in both APC-EpCAM and 488-EGFR. All fluorescence images were taken at a fixed exposure time. The EM gain for each cell and the fluorescence intensity was normalized in order to compare fluorescence images on the plasmonic chip and on the glass slides. Bright-field images were recorded without EM gain.
8
Acridine Orange Fluorescence Assay for Autophagy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze autophagy, cells were reacted with acridine orange (Invitrogen) for 15 min and fluorescence was measured using a fluorescence microscope (BH2-RFL-T3; Olympus, Tokyo, Japan) (Farah et al. 2016 (link)). Depending on the acidity, autophagic lysosomes appeared as orange/red fluorescent cytoplasmic vesicles, while the nuclei were stained green.
9
Autophagy Assessment via Acridine Orange
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hesperidin (50 µM) and/or PM2.5 (50 µg/mL)-treated cells were treated with acridine orange (5 µM; Invitrogen) and incubated for 15 min to assess autophagy. Fluorescence was detected using a fluorescence microscope (BH2-RFL-T3; Olympus, Tokyo, Japan).
10
Acridine Orange Staining of Autophagic Lysosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze autophagy, the cells were reacted with acridine orange (Invitrogen) for 15 min and fluorescence was measured using a fluorescence microscope (BH2-RFL-T3; Olympus, Tokyo, Japan). Depending on acidity, autophagic lysosomes appeared as orange/red fluorescent cytoplasmic vesicles, while the nuclei were stained green [27 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!