Distal colon segments were fixed in 4% paraformaldehyde in PBS overnight at 4°C, washed in PBS, and flash frozen in isopentane cooled with liquid nitrogen before embedding in tissue freezing medium (Thermo 1437365) and storing at −80°C. Sections (8 μm) were cut and mounted on charged glass slides. Antigen retrieval was then performed by heating to 70° C in 10 mM citrate (pH 6.0) for 40 minutes using a microwave on low power. After cooling, sections were permeabilized and blocked in 5% goat serum and 0.1% Tween-20 in PBS (PBST) for 30 minutes, then incubated in blocking solution containing rabbit anti-NKCC1 (1:500) and mouse anti-BKα (Millipore MABN70, 1:100)30 (link) antibodies overnight at 4°C. The following day, sections were washed in PBST (5 minutes × 5) and incubated for 1 hour at room temperature in blocking solution containing Alexa Fluor 488 goat anti-rabbit (Thermo A11008) and Alexa Fluor 568 goat anti-mouse (Thermo A11031). After washing again in PBST, coverslips were mounted with SlowFade Diamond mountant containing DAPI (Thermo S36973) and sealed with clear nail polish. Images were captured with a Zeiss 710 confocal microscope (Carl Zeiss, NY).
Slowfade diamond mountant
Manufactured by Thermo Fisher Scientific
SlowFade Diamond mountant is a reagent used to mount and preserve fluorescently labeled samples for microscopy. It is designed to reduce photobleaching and maintain the intensity of fluorescent signals over time.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal system, by Zeiss (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
710 confocal microscope, by Zeiss (1 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Mabn70, by Merck Group (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Fluorescently labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
4 protocols using slowfade diamond mountant
1
Immunolocalization of NKCC1 and BKα in Distal Colon
2
Microscopic Cell and Stalk Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were spotted onto 1% agarose pads made in the corresponding growth medium. Phase microscopy was performed on a Nikon TiE inverted microscope equipped with a Prior Lumen 220PRO illumination system, Zyla sCMOS 5.5-megapixel camera, CFI Plan Apochromat 100× oil immersion objective (numerical aperture [NA] of 1.45 and working distance [WD] of 0.13 mm), and NIS Elements software for image acquisition. Cell and stalk dimensions were measured using Morphometrics (41 (link)) and ImageJ v. 1.48q (NIH), respectively. To measure membrane permeability, cells were grown in the presence of 1 µg/ml of propidium iodide. EPS production was assessed as previously described (29 (link)). Briefly, 500 microliters of cells grown in HIGG–1 µM phosphate were collected by centrifugation (14,000 × g, 5 min), and the pellet was resuspended in 30 µl of 0.5× phosphate-buffered saline (PBS). Ten microliters of the cell suspension was mixed with 5 µl of FITC-dextran (10 mg/ml; molecular weight [MW], 2 MDa; Sigma) and 1 µl of SlowFade Diamond mountant (Thermo Scientific). Two microliters of this mixture was spotted onto a glass slide, coverslipped, and sealed with vaseline-lanolin-paraffin (VALAP; 1:1:1) for imaging.
3
Immunostaining of Drosophila Germaria and Guts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Germaria were dissected from 7d old females, and guts from 12-14d old females in PBS. The dissected tissues were fixed in 4% PFA at room temperature for 1 h, rinsed in PBST (PBS with 0.3% Triton X-100) for 3 times, and blocked in PBST with 5% normal goat serum for 1.5 h at room temperature. The tissues were then incubated with primary antibody diluted in PBST with 5% normal goat serum overnight at 4 C. The following primary antibodies were used: anti-b-Gal (1:200, Sigma), anti-pros (1:500, DSHB), anti-vasa (1:10000, a gift from Xiaohang Yang), anti-1B1 (1:40, DSHB), anti-LamC (1:20, DSHB), anti-InRb (Tyr1146) (1:200, CST #3021), and anti-E-Cadherin (1:200, a gift from Hong Luo). After rinsed three times in PBST, the tissues were incubated with appropriate fluorescently labeled secondary antibodies (Thermo) in dark for 1 h. They were then stained with DAPI and washed 3 times with PBST before mounting in SlowFade Diamond mountant (Thermo). Images were collected on the ZEISS LSM880 confocal system.
4
Visualizing Lac Operator Array in U2OS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pCDNA5-GFP plasmid was co-transfected with pDsRed-LacI plasmid into LacO-U2OS cells (with Lac operator array inserted) using Lipofectamine 2000 (Invitrogen, 11,668,019). The medium was changed after 8 h, and the cells were harvested 36 h after transfection. The cells were washed with DPBS for 3 times, fixed in 4% PFA at room temperature for 10 min, and then rinsed in DPBS for 3 times. The cells were stained with DAPI (Sigma, D9542) diluted in PBST (DPBS with 0.05% Triton X-100) for 5 min and washed 3 times with PBST before mounting in SlowFade Diamond mountant (Thermo, S36963). Images were then collected on the Zeiss LSM880 confocal system. The images were analyzed and measured with ZEN 2 blue v2.3 (Zeiss). All experiments were performed three or more times, and representative results were shown.
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!