Double immunofluorescence staining experiments were performed as per methods previously described by our laboratory (22 (link)). Before immunofluorescence staining, frozen temporal cortex tissue sections (6-µm) were warmed at 26˚C for 30 min and fixed in ice-cold acetone or other alternate 4% paraformaldehyde fixatives for 10 min. The sections were blocked in 5% FBS for 60 min and incubated with primary antibodies (Anti-NeuN antibody-Neuronal Marker; 1:100; cat. no. ab177487; Abcam; Anti-GFAP antibody; 1:100; cat. no. ab7260; Abcam; and Cdk5-antibody; 1:50; cat no sc-6247; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The sections were washed and incubated with appropriate secondary antibodies (Alexa Fluor® 594-conjugated goat anti-rabbit IgG H&L; 1:300, cat. no. ab150080; Abcam; and Alexa Fluor® 488-conjugated goat anti-mouse IgG H&L; 1:300, cat. no. ab150113; Abcam) for 1 h at 26˚C. Cell nuclei were stained using 4-diamidino-2-phenylindole (DAPI). Fluorescence microscopy was performed with a ZEISS HB050 inverted microscope (magnification, x40; Carl Zeiss AG) and six views of fields were processed using Image-Pro Plus 7.0 (Media Cybernetics, Inc.).
Hb050 inverted microscope
Manufactured by Zeiss
Sourced in Germany
The ZEISS HB050 is an inverted microscope designed for routine observation and imaging of samples. It features a robust and ergonomic design with a compact footprint. The microscope is equipped with a high-quality optical system that delivers clear and detailed images. The HB050 is a versatile instrument suitable for a wide range of applications in various fields of research and diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Tunel detection kit, by Roche (3 mentions)
Image pro plus 7, by Media Cybernetics (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg h l, by Abcam (1 mentions)
Anti gfap antibody, by Abcam (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Tunel kit, by Beyotime (1 mentions)
6 protocols using hb050 inverted microscope
1
Double Immunofluorescence Staining of Brain Tissue
2
Immunofluorescence Staining of NLRP3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, GAS muscle sections (6 μm thickness) or C2C12 myotubes were incubated with primary antibody against NLRP3 (Cell Signaling Technology) overnight at 4 °C. The sections were washed three times with PBS and incubated with secondary antibodies (Alexa FluorTM 594-conjugated, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature and then counterstained with myosin (Alexa FluorTM 488-conjugated, Thermo Fisher Scientific) overnight at 4 °C. At last, they were incubated with 4,6-diamidino-2-phenylindole (DAPI, Millipore Sigma, Burlington, MA, USA) for 4 min at room temperature. Finally, a Zeiss HB050 inverted microscope (Carl Zeiss, Oberkochen, Germany) was used to examine the fluorescence.
3
Quantifying Apoptosis via TUNEL Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apoptotic cells were determined using a TUNEL detection kit (Roche Inc, Indianapolis, IN, USA). The sections were incubated at 37°C with TUNEL reaction fluid for 60 minutes. The slides were washed three times with PBS for 45 minutes, followed by blocking with 10% goat serum in 0.1 mol/L Tris for 15 minutes. The slides were covered with slide glass and mounting medium after three washes. DNA was fixed with streptavidin‐HRP peroxidase (1:40 dilution) and dyed with DAB Chromogen. The apoptotic cells were atrophied with a crimped brown nucleus according to the microscopic analysis (ZEISS HB050 inverted microscope; Carl Zeiss, Thornwood, NY, USA). The positive cells were monitored and analysed by two observers blinded to the experiment. TUNEL‐positive cells from each sample were selected for quantification. The average percentage of the four aspects was regarded as the data for analysis.
4
Quantifying Apoptotic Cells Using TUNEL Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The apoptotic cells were conducted by using a TUNEL detection kit (Roche Inc, Indianapolis, USA). The sections were incubated at 37°C with TUNEL reaction fluid for 60 minutes add the digoxigenin‐conjugated dUTP to the 3′‐OH ends of fragmented DNA and washed with PBS three times for 45 minutes followed by blocking with 10% goat serum in 0.1 m Tris for 15 minutes. The slides were covered by microscopic glass with mounting medium after three washes again. DNA was fixed by streptavidin‐ HRP peroxidase (1:40 dilution) and dyeing with DAB Chromogen. The apoptotic cells showed cell atrophy with crimp brown nucleus was performed by ZEISS HB050 inverted microscope. The positive cells were monitored and analysed by two observers blinding to the experiment. TUNEL‐positive cells in four aspects from each sample were elected for quantification. The average percentage of these aspects was confirmed as the ultimately data.
5
Quantifying Brain Cell Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue and cell apoptosis was detected using TUNEL kits (C1091, Beyotime) in strict accordance with the manufacture’s manuals. First, brain tissue sections were dewaxed with xylene, rinsed with alcohols of gradient concentrations, treated with DNase-free proteinase K at 37°C for 20 min, rinsed with phosphate buffered saline (PBS) thrice, and then incubated with 3% H2O2 for 20 min. Endogenous peroxidase was inactivated. Following incubation with TUNEL mixture for 1 h at 37°C, a HB050 inverted microscope (Zeiss, Hamburg, Germany) was introduced for imaging. The extent of brain injury was assessed using an apoptosis index, which was calculated as average percentage of TUNEL-positive cells in six fields of each section.
6
Apoptosis Detection Using TUNEL Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The apoptotic cells were detected using a TUNEL detection kit (Roche Inc., Indianapolis, IN). The sections were incubated at 37°C with the TUNEL reaction fluid for 60 min, then digoxigenin‐conjugated dUTP was added to the 3′‐OH ends of the fragmented DNA. The sections were then washed three times with PBS for 45 min followed by blocking with 10% goat serum in 0.1 mol/L Tris for 15 min. The slides were washed three more times, then coverslipped with mounting medium. DNA was fixed with streptavidin‐ horseradish peroxidase (1:40 dilution) and counterstained with the DAB chromogen. Atrophied apoptotic cells with crimped brown nuclei were visualized under a ZEISS HB050 inverted microscope. Two observers blinded to the experiment assessed the positive cells. TUNEL–positive cells in four regions of the slide were quantified. The average percentage of these regions was calculated and included in the final data.
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!