At the experimental end-point animals were euthanized and tissues were fresh frozen in liquid nitrogen or fixed in 4% paraformaldehyde in PBS overnight. To review histology, slides were stained by haematoxylin and eosin (H&E). Immunohistochemistry (IHC) and immunofluorescence (IF) were performed using standard protocols. Specificity of immunostaining was assessed by incubation in the absence of primary antibody. We used the following primary antibodies: RFP for Strawberry detection (ab34771, 1:400; Abcam), Aquaporin 1 (NB-600–749, 1:500; Novus Biologicals), THP (AF5175, 1:100; R&D), Aquaporin 2 (ab105171, 1:1000; Abcam), Nephrin (AF3159, 1:100; R&D), CD34 (ab8158, 1:50; Abcam), CD73 (AF4488, 1:100; R&D), GFP (ab290, 1:1000; Abcam), HIF2a (NB100-132, 1:150; Novus Biologicals), CA9 (sc-25600, 1:200; Santa Cruz), turbo-RFP (AB234, 1:500; Evrogen) and pS6 (2211, 1:200; Cell Signalling). Secondary antibodies used were conjugated to HRP (IHC) or Alexa-fluor® fluorochromes (IF). Fluorescent images were obtained by confocal laser-scanning microscopy (Leica TCS SP5).
Ab234
Manufactured by Evrogen
The AB234 is a laboratory equipment designed for general scientific applications. It is a multi-purpose device that can perform various functions as required by the user. The core function of the AB234 is to enable precise and consistent measurements or operations within a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions)
Nb 600 749, by Novus Biologicals (1 mentions)
Af3159, by R&D Systems (1 mentions)
Af4488, by R&D Systems (1 mentions)
Sc 25600, by Santa Cruz Biotechnology (1 mentions)
Tcs sp5, by Leica (1 mentions)
Ab34771, by Abcam (1 mentions)
Ab8158, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Nb100 132, by Novus Biologicals (1 mentions)
Westar sun, by Cyanagen (1 mentions)
Precast sds polyacrylamide gel, by Bio-Rad (1 mentions)
Complete proteinase inhibitor, by Roche (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Laser scanning microscope 880, by Zeiss (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Ab144p, by Merck Group (1 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
S11223, by Thermo Fisher Scientific (1 mentions)
4 protocols using ab234
1
Histological and Molecular Characterization
2
Transient Transfection and Western Blot Analysis of JellyOp Variants
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HEK293 cells were transiently transfected with JellyOp-mKate or JellyOp(K72T)-mKate as described above. Two days after transfection, cells were lysed in standard TNE buffer supplemented with cOmplete™ proteinase inhibitor (Roche). Lysate containing 25 ng protein was incubated in Laemmli buffer for 30 min at 37 °C and run on a 4–20% precast SDS-polyacrylamide gel (Bio-Rad). Protein was transferred to a polyvinylidene difluoride membrane (Immobilion). The membrane was blocked and stained in Tris-buffered saline supplemented with 0.1% Tween 20. The blocking solution (30 min) contained 5% non-fat milk. The primary antibody solution (overnight) contained mouse anti-GAPDH (1:4000, Fitzgerald, 10R-G109A) and rabbit anti-tRFP (1:1000, Evrogen, AB234). The secondary antibody solution (45 min) contained anti-mouse HRP (1:3000, Jackson Immuno Research, 115-035-146) and anti-rabbit HRP (1:3000, Jackson Immuno Research, 111-035-144). Membranes were washed for 10 min between and after incubation steps. Stained membranes were developed using Westar Sun (Cyanagen, XLS063.0250) and imaged on a ChemiDoc MP imaging system (Bio-Rad).
3
Immunohistochemistry of Retinal Cryosections
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Immunohistochemistry of cryosections were similar to that described previously46 (link). In brief, retinas or eyecups were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min and cryoprotected in 30% sucrose. Antibodies were diluted in a blocking solution containing 1% Triton-X and 2% donkey serum. Sections were incubated overnight at 4 °C in primary antibody and 2 h in secondary antibody at room temperature. The following antibodies were used: rabbit anti-tRFP (1:1000; Evrogen; AB234) and donkey anti rabbit conjugated to Alexa 488 (1:400; Invitrogen). Nuclei were stained with 10 μg/ml DAPI (Roche). Micrographs were taken on a Zeiss Laser Scanning Microscope 880. Processing of image stacks was done using ImageJ v 2.3 (Rasband WS, NIH, Bethesda, Maryland, USA).
4
Retinal immunohistochemistry for optogenetics
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At the end of the terminal experiment, mice were euthanized and retinas extracted for subsequent immunohistochemistry to confirm retinal expression patterns of the optogenes. Immunohistochemistry of cryosections were similar to that described previously26 (link). In brief, retinas or eyecups were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min. Antibodies were diluted in a blocking solution containing 1% Triton-X and 2% donkey serum. Sections were incubated overnight at 4 °C in primary antibody and 2 h in secondary antibody at room temperature. The following primary antibodies were used: rabbit anti-tRFP (1:1000; Evrogen; AB234), rabbit anti-melanopsin (1:1000; Advanced Targeting Systems; AB-N39), goat anti-ChAT (1:100; Millipore; AB144P) and mouse anti-Goα (1:1000; Millipore; mab3073). Secondary antibodies were always from donkey and either conjugated to Alexa 488 or Alexa 594 (1:400; Invitrogen). Alexa 488 conjugated to streptavidin was used to visualize cells injected with biocytin during patch-clamp experiments (1:400; Invitrogen; S-11223). Nuclei were stained with 10 μg/ml DAPI (Roche). Micrographs were taken on a Zeiss LSM 880. Processing of image stacks was done using ImageJ (Rasband WS, United States National Institutes of Health, Bethesda, Maryland, US).
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