Cells (2 × 104/well) were seeded in 8-well chamber slides (ibidi GmbH). After 24 h recovery, cells were treated with indicated drug concentrations and fixed with 4% paraformaldehyde for 15 min at room temperature and (after washing with PBS) blocked and permeabilized with 5% FCS, 0.3% Triton X-100 in PBS for 1 h. The primary antibody CHOP (Cell Signaling Technology) was added 1:3200 in 1% BSA and 0.3% Triton X-100 in PBS overnight at 4 °C. After washing with PBS, the cells were incubated with anti-mouse secondary antibody conjugated to AlexaFluor488 (Thermo Fisher, 1:500 in 1% BSA and 0.3% Triton X-100 in PBS) for 1 h. Cells were again washed and counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; 1 µg/ml) and wheat germ agglutinin (WGA, 10 µg/ml, Vector Laboratories, CA, USA) in PBS for 10 min. The dyes were removed, and the cells mounted in Vectashield mounting medium (Vector Laboratories, CA, USA) with a coverslip. Images were taken with a Zeiss LSM 700 Olympus (Carl Zeiss AG, Oberkochen, Germany) confocal microscope and CHOP fluorescence intensities per nucleus were measured using ImageJ.
4 6 diamidine 2 phenylindole dihydrochloride dapi
Manufactured by Vector Laboratories
Sourced in United States
4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) is a fluorescent dye used in molecular biology and fluorescence microscopy. It binds to DNA and emits blue fluorescence when excited by ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
Fitc psa, by Merck Group (2 mentions)
Wheat germ agglutinin (wga), by Vector Laboratories (1 mentions)
8 well chamber slide, by Ibidi (1 mentions)
Anti mouse secondary antibody conjugated to alexafluor 488, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Calcium ionophore a23187, by Merck Group (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Fitc conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluoro jade b, by Merck Group (1 mentions)
Fv10i, by Olympus (1 mentions)
Fluoview fv10i, by Olympus (1 mentions)
Vectashield h 100 mounting medium, by Vector Laboratories (1 mentions)
6 protocols using 4 6 diamidine 2 phenylindole dihydrochloride dapi
1
CHOP Expression Analysis in Cells
2
Acrosome Reaction Induction and Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The induction of the acrosome reaction was performed by 10 µM of calcium ionophore A23187 (Sigma-Aldrich) and 2 mM of calcium chloride (Panreac Química S.L.U, Barcelona, Spain) for one hour at 37 °C and 5.5% (v/v) CO2, following previous protocols of our group (Sáez-Espinosa et al., 2020). Only calcium chloride was added to the controls to assess spontaneous acrosome reaction.
To assess the acrosomal status, 5 µL of each physiological condition (controls and induced samples) were placed on coverslips and fixed in methanol for 30 min. After the smear was dry, cells were washed three times in PBS and unspecific bindings were blocked using 2% (w/v) BSA-PBS for 30 min. The smears were then incubated in the dark with Pisum sativum agglutinin lectin conjugated with fluorescein-5-isothiocyanate (PSA-FITC, Sigma-Aldrich) at a concentration of 50 µg/mL for 30 min. After three washes in PBS, samples were mounted using Vectashield and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Vector Laboratories, Burlingame, CA, USA). DAPI was used to detect the nucleus of the cells and the whole process was conducted at room temperature (Sáez-Espinosa et al., 2020).
To assess the acrosomal status, 5 µL of each physiological condition (controls and induced samples) were placed on coverslips and fixed in methanol for 30 min. After the smear was dry, cells were washed three times in PBS and unspecific bindings were blocked using 2% (w/v) BSA-PBS for 30 min. The smears were then incubated in the dark with Pisum sativum agglutinin lectin conjugated with fluorescein-5-isothiocyanate (PSA-FITC, Sigma-Aldrich) at a concentration of 50 µg/mL for 30 min. After three washes in PBS, samples were mounted using Vectashield and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Vector Laboratories, Burlingame, CA, USA). DAPI was used to detect the nucleus of the cells and the whole process was conducted at room temperature (Sáez-Espinosa et al., 2020).
3
Nanocomplex Cellular Uptake Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The intracellular uptake of nanocomplexes with or without competitor was analyzed by confocal microscopy imaging. Briefly, MCF-7 or PC-3 cells (100 000 cells/well) were seeded on glass coverslips placed in a 12-well culture plate. Cells were incubated overnight and incubated for 30 min with pre-treated serum-free medium with or without 5 mmol/L PBA-NH2 as a competitor. Then, the medium was replaced with fresh serum-free medium containing 2 μmol/L DOX equivalent of pPBA or nanocomplexes 1, 2, and 4 and further incubated for 4 h. To observe the localization of pPBA, FCR648-labeled pPBA was incorporated and diluted with pPBA to control the fluorescence intensity. Cells were washed with DPBS and fixed immediately with 10% neutrally buffered formalin at 4 °C overnight. Cells on the coverslips were mounted with Vectashield anti-fade mounting medium containing 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) (Vector Labs). The intracellular fluorescence of pPBA-FCR648 and DOX was observed by CLSM at excitation/emission wavelengths of 633/647 and 488/530 nm, respectively.
4
Immunofluorescence Analysis of Huntingtin and TUNEL
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 48 h incubation with beditin, the cells (n = 4 per treatment condition) were washed with phosphate buffered saline (PBS), fixed with 4% formaldehyde for 10 min at room temperature (RT), and again washed with PBS for 10 min. Incubation with rabbit monoclonal anti-HTT (D7F7, 1:800, Cell Signaling, Danvers, MA, USA) primary antibodies was performed at room temperature (RT) for 1 h. For TUNEL staining, the In Situ Cell Death Detection Kit, Fluorescein (Cat.# 11684795910, Merck, Darmstadt, Germany) was used according to the manufacturer’s protocol. After washing twice with PBS, the cells were incubated in the dark for 1 h at RT with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:500) from Dianova (Jackson Immunoresearch, West Grove, PA, USA). The cells were then washed with PBS containing 0.05% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), mounted with Vectashield Mounting Medium containing 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Vector Laboratories, Burlingame, CA, USA), and evaluated by fluorescence microscopy with an Olympus BX51 and analySIS Software (Olympus, Hamburg, Germany). Quantification of TUNEL and HTT-positive cells was performed from 8–9 images per cell treatment condition.
5
Fluoro-Jade B Staining for Neurodegeneration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To identify degeneration, FJB staining was performed (29 (link)). Each section of the MOB was mounted on gelatin-coated slides and air-dried overnight. The slides were immersed in 1% NaOH in 80% alcohol for 5 min, 2 min in 70% alcohol, and 2 min in distilled water. Then, the slides were transferred to a 0.06% potassium permanganate solution for 10 minutes. The slides were washed in distilled water for 2 minutes. The staining solution was prepared from a 0.01% stock solution of fluoro-jade b (Millipore, Burlington, MA, USA) in 0.1% acetic acid. The slides were incubated in a final concentration of 0.0004% for 20 min, then dried for 1 hour. After drying, the slides were rinsed 1 min in xylene. The slides were mounted in mounting medium 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Vector Laboratories, Burlingame, CA, USA). All images were captured using a model Fluoview FV10i and FV10i software (Olympus, Japan).
6
Acrosomal Integrity Evaluation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After capacitation, the acrosomal integrity was verified fixing 5µl of sample on coverslips with methanol for 30 minutes. After washing, the samples were incubated in dark with Pisum sativum agglutinin conjugated with fluorescein-5-isothiocyanate J o u r n a l P r e -p r o o f (PSA-FITC, Sigma-Aldrich, Inc.) at a final concentration of 50 µg/mL for 30 minutes as previously reported (Cross et al., 1986) (link). After three washes in PBS, samples were mounted using Vectashield® H-100 mounting medium containing 4', 6-diamidine-2'phenylindole dihydrochloride (DAPI, Vector Laboratories, Inc.). Appropriate negative control experiments were performed.
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!