Frozen tissue sections were rehydrated and blocked [5% goat serum (Sigma-Aldrich), 1% BSA, 1.5 M Tris HCl] for 30 min, as previously described (Seifert et al. 2016 ). Anti-γδ TCR (Biolegend), anti-α-SMA, and anti-Cytokeratin 19 (both abcam) were applied at 4 °C overnight. Secondary antibodies against Mouse IgG labeled with Alexa Flour 633, Rabbit IgG labeled with Alexa Fluor 488, and Guinea Pig IgG labeled with Alexa Fluor 568 (all Thermofisher) were used. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Vector Labs) and embedded in Faramount Mounting Medium (Agilent Dako). Images were acquired on a confocal Leica SP5 MP. For immunohistochemistry, Anti-γδ TCR was applied for 12 h, followed by incubation with secondary antibodies for 30 min. Purified Mouse IgG1 was used as isotype control. ImmPACT™ DAB Peroxidase (Vector Labs) was used according to the manufacturer’s instructions. Slides were imaged on Invitrogen EVOS FL Auto Imaging System (Thermo Fisher Scientific). Quantification was performed by assessing ten hotspots as high-power fields (HPF; 20 ×) per slide.
Invitrogen evos fl auto imaging system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Invitrogen EVOS FL Auto Imaging System is a compact, automated, and fluorescence microscope designed for live-cell imaging. The system features a motorized stage, autofocus capabilities, and multiple LED light sources for a range of fluorescence applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti γδtcr, by BioLegend (1 mentions)
Immpact dab peroxidase, by Vector Laboratories (1 mentions)
Sp5 mp, by Leica (1 mentions)
Faramount mounting medium, by Agilent Technologies (1 mentions)
Anti α sma, by Abcam (1 mentions)
Anti cytokeratin 19, by Abcam (1 mentions)
Goat serum, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Dig oligonucleotide tailing kit, by Roche (1 mentions)
Pgem t vector, by Promega (1 mentions)
6 protocols using invitrogen evos fl auto imaging system
1
Multicolor Immunofluorescence and IHC Analysis
2
In situ Hybridization and Immunodetection of ZmNF-YA8
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In situ hybridization and immunological detection were performed as described by Zhang et al. (Zhang et al., 2022b (link)). In brief, a 120-bp fragment, corresponding to the coding region of ZmNF-YA8, was PCR amplified and cloned into the Promega pGEM-T vector (Cat No. A1360, Promega, Madison, WI, USA). Following sequence verification, PGEM-T-ZmNF-YA8 was linearized with SphI and SalI, which was used as the DNA template for in vitro transcription using a DIG Oligonucleotide Tailing Kit (Cat No. 3353583910, Roche Diagnostics, Basel, Switzerland), which contains T7 and SP6 RNA polymerases and digoxin (DIG)-labeled UTP, generating DIG-labeled sense and antisense RNA probes (Table S1
3
Doublecortin Immunohistochemistry of Brain Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Post-fixed brains were incubated with 30% sucrose in PBS for 1 d at 4 °C, which was repeated once with 30% sucrose in PBS solution. The brains were frozen using dry CO2 ice powder and sliced into 30-μm sagittal sections before being transferred to gelatin-coated slides [48 (link)]. At room temperature, the sections were incubated with 5% bovine calf serum (1 h) and, subsequently, with doublecortin primary antibody (1:100, 2 h), They were rinsed in PBS twice for 15 min each, followed by incubation with Alexa Fluor 488 secondary antibody (Ex/Em = 490/525 nm, 1:200, 2 h) and two additional PBS rinses for 15 min each. The samples were topped in Fluoromount™ Aqueous Mounting Medium and covered with glass coverslips for microscopic imaging. The data were collected using the Invitrogen EVOS FL Auto Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) with a 30× objective. The number of doublecortin-positive stained neurons was counted in the same area of three slides per animal and three animals per group.
4
Photothermal Therapy of U87 Spheroids
5
Doublecortin Immunofluorescence in Brain Slices
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Post-fixed brain hemispheres were submerged in 15% sucrose for 12 h and subsequently with 30% sucrose for another 12 h at 4 °C. The brains were frozen using dry CO2 ice and sliced into 30 μm sagittal sections. At room temperature, sections were blocked with 6% bovine calf serum and incubated with doublecortin primary antibody (1:200 in cell staining buffer, 2 h), rinsed in cell staining buffer twice for 15 min each, and then incubated with Alexa Fluor 488 secondary antibody (Ex/Em = 490/525 nm, 1:200 in cell staining buffer, 2 h) and two additional rinses in staining buffer for 15 min each. Samples were submerged in Fluoromount™ Aqueous Mounting Medium and shielded with glass coverslips for microscopic imaging. The images were photographed using the Invitrogen EVOS FL Auto Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) with a 20× objective. Cells were counted within an identical area of 100 µm wide across all tissue sections, as shown in the images.
6
MWCNT-Induced Vascular Disruption in HBMECs
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!