Cells were grown to 60–70% confluence on glass coverslips in 6-well plates and treated with IP6 for 16 h. After treatment, cells were washed with phosphate-buffered saline (PBS), fixed with acetone:methanol (–20°C, 10 min), and blocked in 10% nonimmune goat serum (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. LC3-II (NOVUS Biologicals, Littleton, CO, USA) antibody was applied (1 h), followed by incubation (1 h) with Alexa Fluor 594 F(ab’)2 fragment of goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA). Nuclei were stained with Hoechst. Then, the slides were assessed using a fluorescent microscope.
Nonimmune goat serum
Manufactured by Merck Group
Sourced in United States
Nonimmune goat serum is a laboratory reagent used as a general blocking and dilution agent in various immunological and biochemical assays. It is derived from the blood serum of healthy goats that have not been intentionally immunized, providing a source of proteins and other biomolecules without antibodies or immune factors.
Automatically generated - may contain errors
Lab products found in correlation
Lc3 2, by Novus Biologicals (1 mentions)
Alexa fluor 594 f ab 2 fragment of goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Fluorescent microplate reader, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by Jackson ImmunoResearch (1 mentions)
Fluoview 1000 laser scanning confocal microscope, by Olympus (1 mentions)
Eukitt, by Merck Group (1 mentions)
5 protocols using nonimmune goat serum
1
Immunofluorescence Assay for LC3-II
2
NF-κB p65 Immunofluorescence in ARPE-19 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After we allowed ARPE-19 cells to grow in high-glucose medium (25 mM) for three days, the cells were fixed with cold methanol, blocked with 25% nonimmune goat serum (Sigma-Aldrich), incubated with the mouse antihuman NF-κB p65 antibody (1:100 diluted in PBS) covering the cell surface for 1 h at 37 °C, and then washed three times with PBS. Subsequently, fluorescein isothiocyanate-conjugated goat antimouse IgG (1:200 dilution) was applied for 1 h. The samples were washed three times with PBS (Jackson ImmunoResearch, West Grove, PA) and placed in a blocking solution at 25 °C for 3 h. All steps were performed according to the manufacturer’s instructions. Nuclei were counterstained with DAPI. The fluorescence intensity was measured using a fluorescent microplate reader (Bio-Rad Laboratories), and related images were captured.
3
Immunofluorescence Analysis of PSD95 in Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neurons were fixed by addition of one culture volume of 4% paraformaldehyde and 4% sucrose for 10 minutes at room temperature (RT), then washed with phosphate-buffered saline (PBS), blocked in PBS with 5% non-immune goat serum (Sigma), and probed for PSD95 following standard procedures. Specificity of secondary antibodies was confirmed in control samples without primary antibody. Images were obtained on an Olympus FluoView 1000 Confocal Laser Scanning Microscope (LSM) using a 40x oil immersion lens. A stack of optical sections with 1024 × 1024 resolution at 1-μm intervals through each neuron was obtained and then flattened into a maximum intensity projection. Analysis was performed on an Apple Macintosh notebook using the National Institutes of Health ImageJ Program. Immunofluorescence images of related conditions were matched for laser power, gain, and offset.
4
Immunostaining of Fixed Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neurons were washed in phosphate-buffered saline (PBS) and then fixed in either 4% formaldehyde/4% sucrose in PBS for 10 minutes at room temperature followed by incubation in methanol prechilled to −20°C for 10 minutes at 4°C or in methanol (−20°C) only for 20 minutes at 4°C. Fixed neurons were then permeabilized and blocked simultaneously (2% Non-Immune Goat Serum; Sigma, and 0.2% Triton X-100) before incubation in primary antibodies overnight and subsequent incubation with secondary antibodies the next day [20] . In the green/purple color scheme, colocalization is indicated by white overlap.
5
Immunohistochemical Analysis of Uterine Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Uterine sections (5 μm in thickness) were deparaffinized and dehydrated in graded ethanol. BrdU incorporation to detect cells in the S phase of the cell cycle was evaluated as previously described [34] . Endogenous peroxidase activity and nonspecific binding sites were blocked. Primary antibodies against the proliferation marker p63, steroid receptors, basal and luminal cytokeratins (CK), vimentin and smooth muscle -actin (-SMA) were incubated overnight at 4ºC (Table 1). The reactions were developed using a streptavidin-biotin peroxidase method and diaminobenzidine (Sigma-Aldrich). Samples A c c e p t e d M a n u s c r i p t 10 were mounted with permanent mounting medium (Eukitt, Sigma-Aldrich). Each immunohistochemical run included negative controls in which the primary antibody was replaced by non-immune goat serum (Sigma-Aldrich). Negative controls for BrdU immunodetection were samples from rats that did not receive BrdU.
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!