Western blot analyses were performed on L4/5 spinal ipsilateral dorsal quadrants, as previously described.23 (link),26 (link) Primary antibodies and dilution ratios used were: rabbit P2X4R 1:400 (Alomone Labs, Jerusalem, Israel), rabbit p38 1:1000 (Cell Signaling, Danvers, MA), rabbit phospho-p38 1:1000 (Cell Signaling), rabbit p65/NFκB 1:500 (Merck Millipore, Billerica, MA), rabbit NLRP3 1:500 (LifeSpan Biosciences, Seattle, WA), guinea pig GLT-1 1:5000 (Merck Millipore), goat Iba1 1:1000 (Abcam, Cambridge, MA), rabbit CD11b 1:500 (Abcam), rabbit ATF3 1:1000 (Santa Cruz Biotechnology, Dallas, TX). Mouse β actin 1:100,000 (Sigma) was used against loading control protein. Secondary antibodies used were: Goat anti-mouse IRDye 680RD 1:15,000 (LI-COR Biosciences, Lincoln, NE), goat anti-rabbit IRDye 800CW 1:15,000 (LI-COR Biosciences), donkey anti-guinea pig IRDye 800CW 1:15,000 (LI-COR Biosciences), and donkey anti-goat IRDye 800CW 1:15,000 (LI-COR Biosciences). Bands were quantified using Image Studio (LI-COR Biosciences).
Donkey anti guinea pig irdye 800cw
Manufactured by LI COR
The Donkey anti-guinea pig IRDye 800CW is a secondary antibody labeled with the near-infrared fluorescent dye IRDye 800CW. This product is designed for use in immunodetection applications where the detection of guinea pig primary antibodies is required.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Irdye 680rd goat anti mouse, by LI COR (1 mentions)
Mouse β actin, by Merck Group (1 mentions)
Rabbit p38, by Cell Signaling Technology (1 mentions)
Irdye 800cw donkey anti goat, by LI COR (1 mentions)
Goat iba 1, by Abcam (1 mentions)
Image studio, by LI COR (1 mentions)
Ab9483, by Abcam (1 mentions)
Ab18039, by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ab32532, by Abcam (1 mentions)
Irdye 680rd donkey anti rabbit, by LI COR (1 mentions)
Anti mouse cy5, by Jackson ImmunoResearch (1 mentions)
Alexa 488 anti rabbit, by Jackson ImmunoResearch (1 mentions)
Anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Odyssey device, by LI COR (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Mouse anti actin, by Merck Group (1 mentions)
Guinea pig anti vglut1, by Merck Group (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
4 protocols using donkey anti guinea pig irdye 800cw
1
Spinal Protein Expression Analysis
2
Tissue Blot Immunostaining of SMC6 and SMC5
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A custom-made human tissue blot was received from ProSci (Poway) containing 15 μg of total cellular proteins from testis, ovary, spleen, skeletal muscle, small intestine, liver, skin, and brain. The staining protocol was performed as described previously [32 (link)] using guinea pig anti-SMC6 (GP anti-SMC6; 1:200; custom made), rabbit anti-SMC6 (1:1000; ab18039; Abcam), and rabbit anti-SMC5 (1:200; sc-134544; Santa Cruz) as primary antibodies. As secondary antibodies, goat anti-rabbit and donkey anti-guinea pig IRDye 800CW (1:15 000; LI-COR Biosciences) were used. As a loading control, a primary goat anti-GAPDH antibody (1:1000; ab9483; Abcam) was used. Image acquisition and quantification were done with the Odyssey Infrared Imaging System (LI-COR Biosciences).
3
Immunocytochemistry and Western Blot Protocols
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Primary antibodies that were used in immunocytochemistry (ICC) and live antibody-uptake are as follows: rabbit antibody against synaptotagmin 1 (Syt1) (mouse cortical cultures, Oyster550-labelled, 1:100, #105103C3, Synaptic Systems), VGLUT1 (rat cortical cultures, 1:1000, #135303, Synaptic Systems), pSer133 of CREB (1:650, #9198 L, CST), mouse antibody against Syt1 (rat cortical cultures, Oyster550-labelled, 1:250, #105311C3, Synaptic Systems) and against VGLUT1 (mouse cultures, 1:250 #135311, Synaptic Systems). Primary antibodies that were used in western blotting are: guinea pig antibody against synapsin 1 (Syn1, 1:1000, #106104, Synaptic System), rabbit primary antibodies against pSer553Syn1 site (human and mouse pSer553 corresponds to pSer551 in rat Syn1, 1:1000, #ab32532, Abcam) and against pSer62Syn1 site (1:1000, #PA5-38336, Thermo Fisher). All the fluorescent secondary antibodies used in ICC were purchased from Jackson Immunoresearch and are as follows: anti-rabbit Alexa 488 (1:1000, #711545152), anti-mouse Cy5 (1:1000, #715175150) and anti-rabbit Cy3 (1:500 for mouse cultures, #711165152). For fluorescent detection of WB, IRDye 800CW donkey anti-guinea pig (#926-32411) and IRDye 680RD donkey anti-rabbit (# 926-6807) from Li-COR were used.
4
Quantitative Immunoblotting of VGLUT1
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Two days after transfection, the HEK293T cells were lysed for 30 min at 4°C in buffer containing 25 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA supplemented with complete protease inhibitor (Roche), the extract sedimented for 20 mins at 14000 g at 4°C and the pellet discarded. Five mg supernatant protein was separated by electrophoresis through 10% SDS-polyacrylamide and transferred to a nitrocellulose membrane. The membrane was blocked in 150 mM NaCl with 50 mM Tris, pH 7.4 (TBS) and 5% dry milk, incubated with guinea pig anti-VGLUT1 (1:2000, Millipore Sigma) or mouse anti-actin (1:3000, Sigma) in TBS + 0.1% Tween-20 overnight at 4°C, washed three times for 5 min each in TBS + 0.1% Tween-20, incubated for 30 min at room temperature with IRDye 800CW Donkey anti-Guinea Pig or IRDye 800CW Goat anti-Mouse IgG (1:20000, LI-COR) in blocking buffer + 0.1% Tween-20, washed three times for 10 min each in TBS + 0.1% Tween-20 and rinsed in TBS. The membrane was scanned using an Odyssey device (LI-COR) and the resulting images quantified in ImageJ.
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