After the conclusion of experiments, mice were deeply anesthetized with sodium pentobarbital (120 mg/kg) and then perfused transcardially with 20 mL 0.9% NaCl solution followed by 20 mL paraformaldehyde PBS solution (PFA, 4%, w/v). The brains were fixed in PFA solution overnight and sectioned. Slices were washed with PBS, fluorescence-enhanced with the anti-GFP antibody (FITC, ab6662, Abcam, Cambridge, MA) or c-Fos antibody (226003, Synaptic Systems, Germany) then Cy5 goat-anti-rabbit IgG (111-175-144, Jackson), incubated with DAPI (C1002, 1:1000, Beyotime, Shanghai, China), and later mounted with DAPI. Fluorescence images were then obtained with an epifluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan) with a 10X (CFI Plan Apo Lambda, 0.45 N.A.) or a 20X (CFI Plan Apo Lambda, 0.75 N.A.) objective lens, and were analyzed with ImageJ software (NIH, U.S.).
Cy5 goat anti rabbit igg
Manufactured by Jackson ImmunoResearch
Sourced in Germany
Cy5 Goat anti-rabbit IgG is a secondary antibody conjugated with the Cy5 fluorescent dye. It is designed to detect and visualize rabbit immunoglobulin G (IgG) in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
C fos antibody, by Cell Signaling Technology (2 mentions)
Streptavidin fitc, by Jackson ImmunoResearch (2 mentions)
Cy5 goat anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Ab6662, by Abcam (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
C1002, by Beyotime (1 mentions)
Alexa fluor 488 anti rat igg, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal antibody to gapdh, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti psmb5, by Abcam (1 mentions)
5 protocols using cy5 goat anti rabbit igg
1
Brain Tissue Fixation and Fluorescence Imaging
2
Affinity-purified antibodies for CLIC protein detection
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AP255 and AP1089, the affinity-purified rabbit polyclonal antibodies to CLIC4 and CLIC1, respectively, have been previously described [17 (link),32 (link)]. Commercial antibodies were as follows: Goat polyclonal antibody to mouse albumin, Bethyl Labs (Montgomery TX) #A90-134; rat monoclonal antibody to CD31 clone MEC13.3, Pharmingen (San Jose, CA) #550274; mouse monoclonal antibody to PCNA, Cell Signaling Technology (Danvers, MA) #2586; rabbit monoclonal antibody to Smad2/3, Cell Signaling Technology #8685; rabbit monoclonal antibody to phospho-Smad2/3, Sigma-Aldrich #SAB4504208; mouse monoclonal antibody to GAPDH, Santa Cruz Biotechnology (Santa Cruz, CA) #SC-32233; goat polyclonal antibody to CLIC5, Santa Cruz Biotechnology #SC-65041 Alexa Fluor 488 anti rat IgG, Life Technologies (Grand Island, NY); Cy5 goat anti rabbit IgG, Jackson Labs (Bar Harbor, ME) #111-175-144; FITC-lectin from Lotus tetragonolobus (LTA), Vector Labs (Burlingame CA) #FL-1321; Alexa Fluor 594-isolectin B4 from Griffonia simpicifolia (IB4), Life Technologies, #I21413; HRP-conjugated secondary antibodies, Thermo Scientific Pierce.
3
Viral Vector Constructs and Immunohistochemistry
4
Viral Vector Constructs and Immunohistochemistry
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AAV2-hSyn-hChR2(H134R)-mCherry (titer: 2.9e12), AAV2-hSyn-mCherry (titer: 4.7e12), and AAV2-hSyn-eNpHR3.0-mCherry (1.5e12) were obtained from the University of North Carolina Vector Core with available stock constructs from the laboratory of K. Deisseroth at Stanford University.
C-Fos antibody (1:5000, Cell Signaling Technology Cat# 4384, RRID:AB_2106617) was a rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids near the carboxy-terminus of human c-Fos protein. TH antibody (1:250, Millipore Cat# AB152, RRID:AB_390204) was a rabbit polyclonal antibody raised against denatured tyrosine hydroxylase from rat pheochro-mocytoma. NeuN antibody (1:1000, EMD Millipore Cat# MAB377, RRID:AB_2298772) was a mouse monoclonal antibody, clone A60, selected from immunoglobulins formed against purified cell nuclei from mouse brain. Secondary antibodies used were purchased from Jackson Immuno Research (Westgrove, PA): Cy5 Goat anti-rabbit IgG (1:500, Cat# 111-175-144, RRID:AB_2338013), Alexafluor 594 Donkey anti-rabbit IgG (1:500, Cat# 711-585-152, RRID:AB_2340621), Cy5 Goat anti-mouse IgG (1:500, Cat# 115-175-146, RRID:AB_2338713), and FITC streptavidin (1:200, Cat# 016-010-084, RRID:AB_2337236).
C-Fos antibody (1:5000, Cell Signaling Technology Cat# 4384, RRID:AB_2106617) was a rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids near the carboxy-terminus of human c-Fos protein. TH antibody (1:250, Millipore Cat# AB152, RRID:AB_390204) was a rabbit polyclonal antibody raised against denatured tyrosine hydroxylase from rat pheochro-mocytoma. NeuN antibody (1:1000, EMD Millipore Cat# MAB377, RRID:AB_2298772) was a mouse monoclonal antibody, clone A60, selected from immunoglobulins formed against purified cell nuclei from mouse brain. Secondary antibodies used were purchased from Jackson Immuno Research (Westgrove, PA): Cy5 Goat anti-rabbit IgG (1:500, Cat# 111-175-144, RRID:AB_2338013), Alexafluor 594 Donkey anti-rabbit IgG (1:500, Cat# 711-585-152, RRID:AB_2340621), Cy5 Goat anti-mouse IgG (1:500, Cat# 115-175-146, RRID:AB_2338713), and FITC streptavidin (1:200, Cat# 016-010-084, RRID:AB_2337236).
5
Myocilin-Induced Proteasome and Autophagy
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Tet-on inducible RGC5 cell lines were treated with Dox for 24 h to induce moderate expression of wild-type, P370L, or Q368X myocilin-GFP. The total cell lysate was subjected to Western blotting using rabbit anti-PSMB5 (Abcam, Cambridge, MA), rabbit anti-LC3 (MBL, Farmingdale, NY), or rabbit anti-GAPDH. The level of PSMB5 or LC3 normalized to that of GAPDH in induced cells was compared to that in non-induced control cells.
For immunofluorescence, cells induced with Dox were fixed and stained with rabbit anti-PSMB5 or rabbit anti-LC3. Cy5-goat anti-rabbit IgG (Jackson ImmunoResearch, 1∶200) was used as the secondary antibody.
For immunofluorescence, cells induced with Dox were fixed and stained with rabbit anti-PSMB5 or rabbit anti-LC3. Cy5-goat anti-rabbit IgG (Jackson ImmunoResearch, 1∶200) was used as the secondary antibody.
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