Cells were fixed in 4% paraformadelhyde in PBS for 15 min at RT. After rinsing 2X with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and blocked with 2% BSA in PBS for 1 h. Cells on coverglass were incubated with primary antibody diluted in PBS with 2% BSA at 4°C overnight. Antibodies were used at the following dilutions: PFKL (1:500), G3BP (1:500). Cells were rinsed 3X with PBS containing 0.02% Tween-20 (PBS-T). Secondary antibodies were diluted in PBS with 2% BSA, and cells were incubated at RT for 1 h. The secondary antibodies used were Alexa Fluor 488 goat anti-rabbit IgG and 594 goat anti-mouse IgG (1:500, Invitrogen). After rinsing 3X with PBS-T, cells on the coverglass were mounted on microscope slides with Prolong Gold anti-fade reagent (Invitrogen). Images were taken using a Leica TCS SP5 confocal microscope with a 63x oil immersion objective and processed using Photoshop software (Adobe).
Tcs sp5 confocal microscope
Manufactured by Adobe
Sourced in United States
The TCS SP5 is a confocal microscope. It is designed to capture high-resolution, three-dimensional images of samples. The microscope utilizes laser technology to produce detailed, optical sections of specimens.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Illustrator cc 2018, by Adobe (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Fluoromount mounting medium, by Merck Group (1 mentions)
Cm1850, by Leica (1 mentions)
Las af, by Adobe (1 mentions)
4 protocols using tcs sp5 confocal microscope
1
Immunofluorescence Staining of Cellular Proteins
2
Imaging Native GFP Fluorescence in Brains
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For imaging of native GFP fluorescence, brains were dissected in PBS (1.86 mM NaH2PO4, 8.41 mM Na2HPO4, and 175 mM NaCl) and fixed for 20 min in 4% paraformaldehyde in PBS at 4°C. Brains containing biocytin fills were incubated in 1:200 streptavidin conjugated to Alexa Fluor 568 (Invitrogen) in PBS containing 0.1% Triton X-100 for 48 hr. Images were collected on a Leica TCS SP5 confocal microscope and processed with ImageJ or Adobe Photoshop.
3
Immunolabeling of First Instar Larval Brains
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First instar larval brains were dissected in ice-cold PBS, placed on a 22 × 22 cover slip and fixed in 4% paraformaldehyde for 18 min at room temperature with slow agitation. Fixed brains were washed three times in PBST (PBS 0,3% Triton X-100), for 20 min each, at room temperature with slow agitation. All these steps were performed in a Columbia staining jar. Primary antibodies (see Key resources table) were prepared in PBST and the cover slips with the brains were incubated overnight at 4°C in a humid chamber. Primary antibody solutions were removed and brains were washed again three times in PBST for 20 min each at room temperature with slow agitation. Next, the secondary antibody (see Key resources table) solutions were added and incubated as described. After overnight incubation, secondary antibody solutions were removed and washes with PBST were performed. Brains were mounted in Vectashield (Vector Laboratories) antifade mounting medium. Images were acquired using Leica TCS SP5 confocal microscope and images were assembled using Fiji and Adobe Illustrator CC 2018. DAPI was added together with the secondary antibodies.
4
Immunofluorescence Analysis of SOD1-G93A Rat Tissues
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Primary cells were fixed for 15 min in 4% paraformaldehyde, permeabilized for 5 min in PBS containing 0.1% Triton X-100. The cells were incubated for 2.5 h at 37 °C with the primary antibody and then stained for 1 h with the appropriate secondary antibody. Tissues samples from WT (n = 3), pre-symptomatic (n = 3), and end-stage SOD1-G93A (n = 3) perfused rats were cryoprotected in increasing concentrations of sucrose (10%, 20%, 30%), frozen and cut in 30 μm sections (CM 1850, Leica, Germany). Immunofluorescence analysis was performed in free-floating with sections in 10% normal donkey serum and 0.3% Triton X-100 for 1 h at room temperature and then incubated with the appropriate antibodies in 2% normal donkey serum and 0.3% Triton X-100 for 48 h at 4 °C. Slides were then incubated with appropriate fluorescent-conjugated secondary antibodies in 2% normal donkey serum and 0.3% Triton X-100 for 3 h at room temperature. In both cases, after PBS washes, slides were incubated with 1 μg/mL 6-diamidino-2-phenylindole (DAPI) and cover-slipped with Fluoromount mounting medium (Sigma Aldrich). Immunofluorescence was analyzed by means of a confocal laser scanning microscope. Samples were analyzed with a Leica TCS SP5 confocal microscope and processed using LAS AF and Adobe Photoshop software (Adobe, San Jose, CA, USA).
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