Cells were plated on glass coverslips coated with poly-D-Lysine. 24h after induction of 97QP-GFP, stress granule assembly was stimulated by addition of 0.5 mM of sodium arsenite for 45 min at 37°C, 5% CO2. After stress, cells were washed three times with PBS and fixed with 4% PFA in PBS for 10 min at room temperature, permeabilized with 0.5% Triton in PBS for 5 min at room temperature, washed with PBS and incubated with blocking buffer (4% BSA in PBS) for 1h at room temperature. Primary antibodies were diluted in blocking buffer and incubated for 1 h at room temperature. To monitor stress granule formation, goat anti-TIA-1 (Santa-Cruz, Dallas, TX; SC-1751; 1/100) and mouse anti-G3BP (BD Biosciences, Franklin Lakes, NJ; #611126; 1/1000) were used. After washing with 0.1% Tween in PBS, cells were incubated with IgG (H+L) secondary antibodies donkey anti-goat Alexa 647 or donkey anti-mouse Cy3 (Thermo Fisher Scientific, 1/500) for 45 min. Coverslips were mounted in Prolong Gold Antifade mounting medium (Thermo Fisher Scientific).
Donkey anti mouse cy3
Manufactured by Thermo Fisher Scientific
Donkey anti-mouse Cy3 is a secondary antibody conjugated with the fluorescent dye Cy3. It is specifically designed to detect and visualize mouse primary antibodies in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (6 mentions)
A11122, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit cy2, by Thermo Fisher Scientific (2 mentions)
T1299, by Merck Group (2 mentions)
Mouse anti g3bp, by BD (1 mentions)
Donkey anti goat alexa 647, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Goat anti tia1, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
4 protocols using donkey anti mouse cy3
1
Visualizing Stress Granule Formation
2
Immunofluorescence Imaging of C. elegans
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One day old worms were microirradiated, recovered, and dissected in M9 solution on a coverslip and transferred onto positively charged slides, then placed onto metal blocks in dry ice. Worms were kept in the dark as much as possible throughout this protocol to avoid bleaching. Antibody staining was performed by the following procedure: wash in -20 ⸰ C methanol for 1 minute, 4% PFA for 30 minutes, wash in 1xPBST for 10 minutes, block in 0.5% BSA in 1xPBST for 1-2 hours, then incubated with the primary antibody overnight at room temperature. Following primary antibody incubation, slides were washed in 1xPBST 1-3 times for 10 minutes each, then incubated with the secondary antibody for 2 hours at room temperature in the dark, followed by 1xPBST wash for 10 minutes, 10 minute staining in DAPI (1:10,000 of 5mg/ml stock in 1xPBST), and a wash in 1xPBST for 10 minutes-1 hour. All antibodies used were diluted in 1xPBST. For the SYP images presented in Figure 8A, no microirradiation was performed prior to dissection and staining. Primary antibodies used were: rabbit anti-RAD-51 (1:30,000), mouse anti-FLAG (1:500; Sigma F1804), rabbit anti-OLLAS (1:1,000; Genscript #A01658), goat anti-SYP-1 (1:500).
Secondary antibodies used were: goat anti-rabbit Alexa Fluor 555 (1:500; Invitrogen), donkey anti-rabbit Alexa Fluor 488 (1:500; Thermo), and donkey anti-mouse Cy3 (1:500).
Secondary antibodies used were: goat anti-rabbit Alexa Fluor 555 (1:500; Invitrogen), donkey anti-rabbit Alexa Fluor 488 (1:500; Thermo), and donkey anti-mouse Cy3 (1:500).
3
Dual-Labeling Immunohistochemistry Protocol
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Immunohistochemistry was conducted according to published procedures11 (link). For GFP/TH and TMT/TH double-labeling, brain sections were incubated in 1:4000 of anti-TH (T1299, Sigma) 1:2000 of rabbit polyclonal anti-GFP (A11122, Invitrogen), or 1:1000 of rabbit anti-dsRed (632496, Clontech), respectively, in block solution overnight at 4°C. The next day, sections were incubated in 1:500 of donkey anti-rabbit Cy2 (Immuno Research) for anti-GFP together with 1:500 of donkey anti-mouse Cy3 for anti-TH or 1:500 of donkey anti-rabbit Cy3 for anti-dsRed together with 1:500 of donkey anti-mouse Cy2 for anti-TH in PBS for 1 hr. For GFP/Cre double-labeling, 1:2000 of rabbit polyclonal anti-GFP (A11122, Invitrogen), 1:1000 mouse anti-Cre recombinase (MAB3120), 1:500 of donkey anti-rabbit Cy2, and 1:500 of donkey anti-mouse Cy3 were used. All sections were imaged on a LSM 710 confocal microscope (Zeiss) at 10× or 20× magnification. All histological procedures were replicated independently at least 2 times.
4
Dual-Labeling Immunohistochemistry Protocol
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