Brains were cut into coronal sections (40 μm) on a cryostat, placed in 1 ml of anti-freeze solution (40% PBS, 30% ethylene-glycol, 30%, glycerol, v/v) and stored at −20°C until immunohistochemistry. Triple immunostaining was performed on coronal slices with the free-floating method as previously described (Lana et al., 2014 (link)). The following primary antibodies were used a mouse monoclonal anti-neuronal nuclei (NeuN, 1:200; Millipore, Billerica, MA) for neurons; a rabbit polyclonal anti-glial fibrillary acidic protein (GFAP, 1:1,000; DakoCytomation, Glostrup, Denmark) for astrocytes; a rabbit polyclonal anti-IBA1 (1:300, WAKO Pure Chem. Ind., Osaka, Japan) for microglia. Slices were observed under a ZEISS LSM 5 confocal laser scanning microscope. Confocal scans were taken at 0.5 μm z-steps keeping pinhole, contrast, and brightness constant. Semi-automated image analysis was performed using Bitplane IMARIS 7.4 3D image analysis software (Oxford Instruments, Concord, MA) and the 3-D reconstructed images of microglia and astrocyte were using surface function based on previously described (Radford et al., 2015 (link)).
Lsm 5 confocal laser scanning microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 5 is a confocal laser scanning microscope developed by Zeiss. It is designed to capture high-resolution images by scanning samples with a focused laser beam and collecting the emitted light. The microscope uses advanced optical and digital technologies to provide detailed, three-dimensional visualization of samples.
Automatically generated - may contain errors
Lab products found in correlation
Mounting medium, by Beyotime (2 mentions)
Immunol staining blocking buffer, by Beyotime (2 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Mouse anti histone, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Lsm 5 pascal, by Zeiss (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Mounting medium, by Vector Laboratories (1 mentions)
Zen software, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Lsm 5 software version 3, by Zeiss (1 mentions)
Argon krypton laser, by Zeiss (1 mentions)
Rhodamine tritc conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Fluorescein fitc conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated rabbit anti mouse igg, by Abcam (1 mentions)
10 protocols using lsm 5 confocal laser scanning microscope
1
Immunohistochemistry of Neurons, Astrocytes, and Microglia
2
Immunofluorescence Staining of Insect Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were dissected 72 hAH in 3.7% formaldehyde and fixed for 25 minutes at RT. Tissues were washed in 0.4% Triton X-100 in 1X PBS (0.4% PBT), 3X 15 minutes, and blocked in 10% NGS in 0.4% PBT at RT for 1–2 hours. Tissues were incubated in primary antibody in 0.4% PBT at 4°C overnight [1∶100 rat-anti Sima (P. Wappner), 1∶100 rabbit-anti phospho-Mad (E. De Robertis), 1∶100 mouse-anti Histone (Millipore)], then washed extensively in 0.4% PBT and incubated in secondary antibody in 10% NGS +0.4% PBT at 4°C overnight or 2 hours RT [mouse-FITC, rat-FITC, rabbit-Cy3 (Jackson Immunochemicals, PA)]. Tissues were again washed, incubated in 1∶500 TO-PRO3 (TOPRO; Invitrogen, CA) in distilled water for 30–60 minutes at RT (if noted), and mounted in Vectashield (Vector Laboratories, MI). Images were obtained using a Zeiss LSM5 confocal laser scanning microscope.
The procedure for Dilp2 analysis was adapted from [20] (link), and is the same as outlined above but with the following exceptions: 5% BSA in 0.4% PBT was used instead of 10% NGS in 0.4% PBT. Primary antibody was 1∶100 rat-anti Dilp2 (P. Leopold) and secondary antibody was rat-Cy3 (Jackson Immunochemicals, PA).
The procedure for Dilp2 analysis was adapted from [20] (link), and is the same as outlined above but with the following exceptions: 5% BSA in 0.4% PBT was used instead of 10% NGS in 0.4% PBT. Primary antibody was 1∶100 rat-anti Dilp2 (P. Leopold) and secondary antibody was rat-Cy3 (Jackson Immunochemicals, PA).
3
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
When cells were 60% confluent, DMEM medium was discarded. Coverslips for cell culture were washed three times with phosphate-buffered saline, fixed in pre-cooled ethanol (4 °C) for 15 min, and washed again with PBS. The coverslips were treated with 0.5% TritonX-100 at room temperature for 20 min and blocked with 1% BSA at 37 °C for 1 h. The specimens were then incubated with primary antibodies (1:100) at 4 °C overnight followed by incubation with FITC- and TRITC-labeled secondary antibodies (1:100) in the dark at 37 °C for 2 h. The nucleus was stained with 0.5 μg/mL DAPI. Cell fluorescence imaging was examined using a Zeiss LSM5 confocal laser scanning microscope.
4
Apoptosis Quantification via TUNEL and CASP3 Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was performed according to the manufacturer’s instructions of DeadEnd Fluorometric TUNEL system (Promega, G3250). Confocal imaging of apoptotic cells on slides was acquired using a LSM 5 confocal laser scanning microscope (Carl Zeiss MicroImaging, Jena, Germany) using LSM 5 Pascal software version 3.0. For excitation, a 488 nm argon laser was used for fluorescein-12-dUTP (emission collected with BP 520–555 nm filter), and the 405 nm diode laser was used for DAPI (emission collected with BP 415–480 nm filter).
The CASP3 activity assay was performed using the CaspACE™ Assay System, Colorimetric (Promega, G7220) according to the manufacturer’s instructions. The results are presented as the mean CASP3 activity (± SE) of 3 different experiments.
The CASP3 activity assay was performed using the CaspACE™ Assay System, Colorimetric (Promega, G7220) according to the manufacturer’s instructions. The results are presented as the mean CASP3 activity (± SE) of 3 different experiments.
5
Immunofluorescence Staining of Virus-Infected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Virus-infected or plasmid DNA-transfected cells were fixed with 3.7% paraformaldehyde for 15 min before permeabilizing with 0.5% Triton X-100 followed by blocking with 5% FBS. After blocking the cells with 5% FBS for 60 min, the cells were incubated with protein-specific primary antibody followed by fluorophore-conjugated secondary antibodies. Finally, the cells were mounted with mounting medium (VectaShield) containing 4′, 6-diamidino-2-phenylindole (DAPI) and imaged under Zeiss LSM 5 Laser scanning confocal microscope.
6
Immunofluorescence Staining of Cell-Cell Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining was performed as described before [17 (link)]. Briefly, cell monolayers were fixed (3% paraformaldehyde), permeabilized (0.2% Triton X100), and blocked (4% non-fat milk in TBST). Cells were incubated with primary antibodies (mouse monoclonal anti-occludin (1:200 dilution) and rabbit polyclonal anti-ZO-1 (1:400 dilution) antibodies or mouse monoclonal E-cadherin (1:200 dilution) and rabbit polyclonal anti-β-catenin antibodies (1:300 dilution). The initial incubation was followed by incubation with secondary antibodies (AlexaFluor 488-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG antibodies; 1:100 dilutions) for another hour. AlexaFlour-488-conjugated phalloidin was used to stain F-actin. Fluorescence was examined under a Zeiss LSM 5 laser scanning confocal microscope, and images from x-y sections (1 μM) were collected using Zen software (Zeiss, White Plains, NY, USA). Images were stacked using the software Image J 2.1.0 (NIH, Bethesda, MD, USA) and processed by Adobe Photoshop 21.1.0 (Adobe Systems Inc., San Jose, CA, USA).
7
BiFC Assay in Yeast Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The BiFC assay was performed as described earlier [27 (link)]. Briefly, the competent yeast cells were co-transfected with indicated plasmid DNAs according to the polyethylene glycol –Lithium acetate (PEG-LiAc) method (Clontech Laboratories 2009). Co-transformed cells were plated on to a selective drop-out medium supplemented with 2% glucose in the absence of urease and histidine and incubated at 30 °C for 3–5 days. Finally, the colonies were picked, spread and fixed on slides, mounted with a mounting medium containing DAPI (Vectashield) and examined using a Zeiss LSM 5 laser scanning confocal microscope.
8
Visualizing Chloroplast Morphology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CM was conducted with an LSM5 laser scanning confocal microscope equipped with an argon-krypton laser (Carl Zeiss Inc.). A 488 nm excitation line and an AOBS filter-free system were employed to collect emitted light between 498 and 700 nm. The autofluorescence of chlorophyll was used to visualize chloroplast structure. Collections of optical sections through chloroplasts were used to obtain a three-dimensional reconstruction of chloroplast morphology. The reconstructions of chloroplasts were made by LSM 5 software version 3.5 (Carl Zeiss Inc.).
9
Quantifying DNA Damage and Repair Foci
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunfluorescence detection of phospho- H2AX and RAD51 foci was performed to monitor DNA double-strand breaks (DSBs) formation and homologous recombination repair (HRR). Cells cultured on coverslips were treated with 10 nM YM155 and irradiated with a dose of 4 Gy to assure a discrimination of individual nuclear foci in immunofluorescence staining. At indicated time points, the cells were fixed by 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton X-100 for 10 min at 4°C. After blocking with Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 1 h at room temperature, cells were incubated with antibody for phospho-H2AX (Ser139) (Millipore/Upstate, Temecula, CA) and RAD51 (EMD Millipore, Billerica, MA) at 4°C overnight, followed by staining with Fluorescein (FITC)-conjugated goat anti-mouse IgG (Jackson Immunoresearch, PA) and Rhodamine (TRITC)-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, PA) for 1.5 h at room temperature. Finally, the samples were counterstained with 2 μg/ml DAPI and mounted in 3 μl of mounting medium (Beyotime). Three random fields each containing 50 cells were examined at a magnification of ×100 under a Zeiss LSM5 confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). Nuclei containing ≥10 immunoreactive foci were scored as positive for γ-H2AX, and ≥5 foci as positive for RAD51.
10
Quantifying DNA Double-Strand Breaks via γ-H2AX Foci
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence detection of phospho-H2AX foci was performed to monitor formation of DNA double-strand breaks (DSBs). Cells cultured on coverslips were treated with plasma for 20 sec and were irradiated with a dose of 4 Gy to assure a discrimination of individual nuclear foci in immunofluorescence staining. At indicated time-points, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and were permeabilized with 0.1% Triton X-100 for 10 min at 4°C. After blocking with Immunol Staining Blocking Buffer (Beyotime Institute of Biotechnology, Shanghai, China) for 1 h at room temperature, the cells were incubated with antibody for phospho-H2AX (Ser139) (1:1,000 dilution; cat. no. ab2893; Abcam, Cambridge Science Park, Cambridge, UK) at 4°C overnight, followed by staining with fluorescein (FITC)-conjugated rabbit anti-mouse IgG (10 µg/ml dilution; cat. no. 315-005-045; Jackson ImmunoResearch, West Grove, PA, USA) for 1.5 h at room temperature. Finally, the samples were counterstained with 2 µg/ml DAPI and mounted in 3 µl of mounting medium (Beyotime Institute of Biotechnology). Three random fields each containing 50 cells were examined at a magnification of ×100 under a Zeiss LSM5 confocal laser-scanning microscope (CarlZeiss, Jena, Germany). Nuclei containing ≥10 immunoreactive foci were scored as positive for γ-H2AX.
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!