Immunostaining of myofiber preparations was performed as previously described [37 (link)]. Briefly, myofiber preparations were fixed with 4% PFA, for 20 min, permeabilized with PBST (0.3% TritonX in PBS), blocked for 1 hour with PBSTB (5% BSA in PBT) and incubated overnight at 4°C with primary antibodies. The following primary antibodies were used: mouse anti-DHPR (1:200; DHPRa1A; Abcam), mouse anti-α-Actinin (1:100; Sigma), mouse anti-RyR1 (1:100; 34C; DSHB), rabbit anti-Junctin (1:350; gift from Dulhunty lab), mouse anti-PI(3)P (1:100; Echelon Biosciences Inc.), mouse anti-PI(3,4)P2 (1:100; Echelon Biosciences Inc.), mouse anti-PI(4,5)P2 (1:100; Echelon Biosciences Inc.). Alexa Fluor-conjugated secondary antibodies were used at 1:1000 (Invitrogen). Rhodamine phalloidin (Phalloidin 555) was used to visualize filamentous actin (1:300, Molecular Probes). Preparations were mounted with ProLong Gold with DAPI (Invitrogen). Images were acquired with a Nikon Eclipse Ti laser scanning confocal using NIS Elements software (Nikon Corporation, Tokyo, Japan) and only adjusted for brightness and contrast using Adobe Photoshop.
Mouse anti pi 3 4 p2
Manufactured by Echelon Biosciences
Sourced in United States
Mouse anti-PI(3,4)P2 is a monoclonal antibody that specifically binds to phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), a lipid second messenger involved in cellular signaling pathways. This antibody can be used for the detection and quantification of PI(3,4)P2 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dhpra1a, by Abcam (2 mentions)
Rhodamine phalloidin phalloidin 555, by Thermo Fisher Scientific (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Prolong gold with dapi, by Thermo Fisher Scientific (2 mentions)
Mouse anti pi 3 p, by Echelon Biosciences (2 mentions)
Mouse anti pi 4 5 p2, by Echelon Biosciences (2 mentions)
Mouse anti α actinin, by Merck Group (1 mentions)
Nis elements software, by Nikon (1 mentions)
Mouse anti actinin, by Merck Group (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Lysotracker blue, by Thermo Fisher Scientific (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
3 protocols using mouse anti pi 3 4 p2
1
Immunostaining of Myofiber Preparations
2
Immunostaining of Myofiber Preparations
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Immunostaining of myofiber preparations was performed as previously described (Horstick et al., 2013) . Briefly, myofiber preparations were fixed with 4% PFA, for 20 min, permeabilized with PBST (0.3% TritonX in PBS), blocked for 1 hour with PBSTB (5% BSA in PBT) and incubated overnight at 4 o C with primary antibodies. The following primary antibodies were used: mouse anti-DHPR (1:200; DHPRa1A; Abcam), mouse anti--Actinin (1:100; Sigma), mouse anti-RyR1 (1:100; 34C; DSHB), rabbit anti-Junctin (1:350; gift from Dulhunty lab), mouse anti-PI(3)P (1:100; Echelon Biosciences Inc.), mouse anti-PI(3,4)P2 (1:100; Echelon Biosciences Inc.), mouse anti-PI(4,5)P2 (1:100; Echelon Biosciences Inc.). Alexa Fluor-conjugated secondary antibodies were used at 1:1000 (Invitrogen). Rhodamine phalloidin (Phalloidin 555) was used to visualize filamentous actin (1:300, Molecular Probes). Preparations were mounted with ProLong Gold with DAPI (Invitrogen). Images were acquired with a Nikon Eclipse Ti laser scanning confocal using NIS Elements software (company, location) and only adjusted for brightness and contrast using Adobe Photoshop.
3
Immunofluorescence and Lysotracker Microscopy
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The 293T cells were grown in DMEM 10% FBS over coverslips and then fixed in 4% paraformaldehyde for 20 min. Cells were blocked for 1 h in 1% FBS and 0.1% Triton X-100 in PBS and incubated overnight with the primary antibody, mouse anti-PI (3,4) P2 (1:100, # Z-P034B, Echelon Biosciences Inc (Salt Lake city, UT, USA). Anti-rabbit IgG Cy3 (1:200, Jackson Immunoresearch, West Grove, PA, USA) secondary antibodies were used, and DNA was stained with DAPI.
For the Lysotracker assay, U87 and U373 cells were grown in DMEN 10% FBS over coverslips and incubated with LysoTracker Red or LysoTracker Blue (1:500, Invitrogen) for 15 min, washed in PBS, and immediately imaging was done with Leica SP-5 confocal microscope.
For the Lysotracker assay, U87 and U373 cells were grown in DMEN 10% FBS over coverslips and incubated with LysoTracker Red or LysoTracker Blue (1:500, Invitrogen) for 15 min, washed in PBS, and immediately imaging was done with Leica SP-5 confocal microscope.
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