Skin biopsies (3 mm) were obtained, processed and analyzed according to consensus standards (Lauria et al., 2010 (link)). Tissue was placed in Zamboni's fixative (NewcommerSupply, Middleton, WI, USA) for 24hrs at room temperature, rinsed with 0.01M phosphate buffered saline (PBS) and placed in cryoprotectant (20% glycerol in 0.1M Sorrenson's phosphate buffer) for a minimum of 24hrs at 4°C. Serial frozen sections 70μm thick were made. Sections were permeabilized in 0.5% Triton X-100 for 30 minutes at room temperature, incubated in TNB (0.1M Tris HCl, 0.15M NaCl and 0.5% Boehringer milk powder) with 1% Triton X-100 for 2hrs at room temperature, then treated overnight at 4°C with primary mouse antibodies against myelin basic protein (Calbiochem Cat#NE1019-100ul, 1:1000) and anti-human PGP9.5 from rabbits (ABDserotec Cat#7863-0504, 1:1000) diluted in TNB with 0.5 % triton X-100. Sections were washed in 0.01M PBS with 0.5% triton X-100 and fluorescent secondary antibodies FITC-conjugated donkey anti-rabbit and Cy3-conjugated donkey anti-mouse (Jackson ImmunoResearch; 1:500) applied for 2hrs at room temperature. Sections were washed in 0.01M PBS with 0.5% Triton X-100, then 1mM CuSO4 for 10 minutes, and mounted on glass slides with VECTASHIELD Mounting Medium (Vector Labs, Burlingame, CA).
Fitc conjugated donkey anti rabbit
Manufactured by Jackson ImmunoResearch
Sourced in United States
FITC-conjugated donkey anti-rabbit is a secondary antibody used in immunoassays and other applications. It is produced by immunizing donkeys with rabbit IgG and then conjugating the resulting antiserum with the fluorescent dye FITC (fluorescein isothiocyanate).
Automatically generated - may contain errors
Lab products found in correlation
Cy3 conjugated donkey anti mouse, by Jackson ImmunoResearch (4 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Ne1019 100ul, by Bio-Rad (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Cy5 conjugated donkey anti mouse, by Jackson ImmunoResearch (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Ab109320, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Tritc conjugated donkey anti mouse, by Jackson ImmunoResearch (1 mentions)
F1804, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Rabbit anti mouse lyve 1, by Abcam (1 mentions)
Axio imager m1, by Zeiss (1 mentions)
Vectorshield, by Vector Laboratories (1 mentions)
Rat anti dpn, by Abcam (1 mentions)
Cy3 conjugated donkey anti rat, by Jackson ImmunoResearch (1 mentions)
16 protocols using fitc conjugated donkey anti rabbit
1
Immunohistochemical Analysis of Skin Biopsies
2
Immunostaining Protocol for DUX4 and Related Proteins
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Cells were fixed for 10 min with 2% paraformaldehyde (Thermo Scientific) for DUX4/STAT1 and 4% paraformaldehyde for DUX4/IDO1, then permeabilized for 10 min with 0.5% Triton X-100 (Sigma), both at room temperature with gentle shaking. Cells were then blocked for 2 hr with PBS/0.3 M glycine/3% BSA at room temperature with gentle shaking. Primary antibodies were incubated at 4°C overnight at the following concentrations: rabbit anti-IDO1 [D5J4E] 1:100 (Cell Signaling Technology, 86630S, RRID:AB2636818 ), mouse anti-DUX4 [P2G4] 1:250 (Geng et al., 2011 (link)), rabbit anti-DUX4 [E5-5] 1:1000 (Geng et al., 2011 (link)), rabbit anti-DUX4 [E14-3] 1:1000 (Geng et al., 2011 (link)), mouse anti-FLAG [M2] 1:500 (Sigma #F1804, RRID:AB_262044 ), rabbit anti-STAT1 [EPR4407] 1:750 (Abcam #ab109320, RRID:AB_10863383 ), and rabbit anti-pSTAT1 Y701 [58D6] 1:400 (Cell Signaling Technology #9167). Cells were washed three times with 1× PBS containing 3% BSA, then secondary antibodies were incubated for 1 hr at room temperature: FITC-conjugated donkey anti-rabbit (Jackson ImmunoResearch #711-095-152, RRID:AB_2315776 ) or TRITC-conjugated donkey anti-mouse (Jackson ImmunoResearch #715-025-020, RRID:AB_2340764 ). Cells were washed once with 1× PBS containing 3% BSA then stained with DAPI (Sigma) 1:5000 for 10 min at room temperature and visualized.
3
Cornea Immunofluorescent Staining Protocol
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The experiment was performed as described previously.7 (link)–9 (link, link) Briefly, whole-mount full-thickness corneas were harvested at 8 weeks after transplantation and fixed in acetone for immunofluorescent staining. Samples were sequentially incubated with purified rabbit anti-mouse LYVE-1 (Abcam, Cambridge, MA, USA) antibody and goat anti-mouse Itga-9 antibody (R&D Systems, Minneapolis, MN, USA), which were visualized by FITC-conjugated donkey anti-rabbit and Cy3-conjugated donkey anti-goat secondary antibodies (Jackson ImmunoResearch Laboratories), respectively. Samples were covered with Vector Shield mounting medium (Vector Laboratories, Burlingame, CA, USA) and examined by an AxioImager M1 epifluorescence deconvolution microscope with AxioVision 4.8 software (Carl Zeiss AG, Göttingen, Germany).
4
Immunohistochemical Staining of Drosophila Larvae
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Third-instar wandering larvae were dissected in PBS and fixed in 4% formaldehyde in PBS. Samples were washed three times after fixation with PBS containing 0.3% Triton X-100 and transferred to blocking solution (PBS containing 5% normal donkey serum and 0.3% Triton X-100). Specimens were incubated overnight at 4 °C with primary antibodies diluted in blocking solution. Primary antibodies were washed four times with PBS containing 0.3% Triton X-100 before the incubation overnight at 4 °C with secondary antibodies. Then, the samples were washed four times with PBS containing 0.3% Triton X-100. The primary antibodies used were as follows: rabbit anti-PatJ (1:1000), rat anti-Dpn (Abcam, 1:50), and guinea pig anti-L’sc (1:1200). The following secondary antibodies (Jackson) were used at 1:200 dilutions: FITC-conjugated donkey anti-rabbit, Cy3-conjugated donkey anti-rat, and Alexa Fluor 647-conjugated donkey anti-guinea pig. Specimens were mounted with VectaShield mounting media (Vector) and viewed on a Zeiss LSM880 confocal microscope. ZEN software (Zeiss) was used for preparing three-dimensional images.
5
Immunohistochemical Analysis of Skin Biopsies
6
Immunofluorescence Staining of γ-H2AX
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Cells were grown on a glass coverslip, fixed, and permeabilized with 4%PFA/0.5%-Triton X-100 for 20 min at room temperature, then washed with PBS before adding appropriate primary and secondary antibodies. Cells were stained with the following primary antibodies: Rabbit γ-H2AX (cell signaling, # 9718) and the secondary antibodies: FITC-conjugated donkey anti-rabbit (Jackson Immunoresearch, West Grove, PA, USA). DNA was stained using DAPI (4′,6-diamidino-2-phenylindol-dihydrochloride) (Roche, Mannheim, Germany). Slides were examined using an Axio Imager 7.1 microscope (Zeiss, Göttingen, Germany), and images were taken using an Axio Cam MRm camera (Zeiss, Göttingen, Germany).
7
RNA-RNA and RNA-Protein Double Labeling
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The majority of RNA-RNA and RNA-protein double labeling was performed as described in58 (link), with the following modifications. 0.1% Tween-20 and 0.03% Triton X-100 were used for RNA-protein double-labeling. The following antibodies were used: rabbit anti-GFP (1:400, Abcam # 290), mouse anti-S5 (1:2, obtained from Harald Saumweber), Alexa488-conjugated goat anti-mouse (1:000; Invitrogen #A-11001) and FITC-conjugated donkey anti-rabbit (1:1000; Jackson ImmunoResearch #711-095-152). The CD63-GFP expressing fly stock, A17122 (w; spitz-GAL4, tub-GAL80ts; UAS CD63-GFP/SM5 TM6), was provided by Clive Wilson.
8
Visualizing KSHV Protein K15 Localization
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2.5x104 HuAR2T r.KSHV.219 cells were plated on glass coverslips and thirty-six hours later washed with 1xPBS and fixed with 100% ice cold methanol for 20 min at -20°C. Cells were then thoroughly washed with 1xPBS and incubated for one hour in 10% FCS in PBS at 37°C. Coverslips were then incubated with a rat monoclonal antibody to K15 (clone 18E5) for one hour at 37°C, washed 3 times in 1x PBS and incubated for one hour with Cy3-labelled anti-rat secondary antibody (712-165-153 Jackson Immuno Reasearch) for one hour at 37°C. Coverslips were washed, fixed with 4% PFA 20 minutes, PFA was quenched with 1xTBS 10 min at room temperature. Coverslips were then stained as indicated. The following fluorescently labelled secondary antibodies were used: FITC conjugated donkey anti-rabbit (711-095-152 Jackson Immuno Research), Cy5 conjugated goat anti-mouse (115-175-164 Jackson Immuno Research). Images were taken with ZEISS LSM 510 Meta scan head connected to an inverted microscope Axiovert 200M. To analyse the co-localisation the JACOP tool was used and the Pearson’s coefficient (PC) was calculated and shown in Figs 2 and 3A [52 (link)].
9
Antibodies for Retinal Protein Analysis
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The following antibodies were used in this study. Primary antibodies: PhLP1 [19 (link)], Gβ1 [20 (link)], RGS9-1 [21 (link)] and cone arrestin [22 (link)] were made and characterized as described previously by members of our research team. Gαt2 and Gγc [23 (link)] was a generous gift from Dr. Vadim Arshavsky (Duke University). Gαt1 and Gγ1 (Santa Cruz), Gβ3 (Sigma), Gβ5 (Proteintech), and cone M-opsin (Millipore) were from commercial sources. Secondary antibodies: FITC-conjugated donkey anti-rabbit (Jackson ImmunoResearch Laboratories), TRITC-conjugated peanut agglutinin (Vector Laboratories), AF555-conjugated goat anti-rabbit (Life Technologies) were all from commercial sources.
10
Whole-Mount Liver Organoid Staining
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For whole-mount liver organoid staining, organoids were fixed in 4% paraformaldehyde for 30 min at 4°C, washed with PBST (0.1% Tween in PBS) for three times, and permeabilized with PBS containing 0.2% Triton X-100 (Sigma) for 15 min. Organoids were then washed with PBST for three times and blocked by 5% BSA for 1 h at room temperature. Organoids were incubated with primary antibodies at 4°C overnight, washed with PBST for three times, and incubated with secondary antibody and DAPI for 1 h at room temperature. Organoid imaging was performed on confocal microscope (OLYMPUS, FV3000). The following antibodies were used: goat anti-Albumin (Bethyl), rabbit anti-Sox9 (Santa Cruz), mouse anti-E-cadherin (BD), mouse anti-Ki67 (BD), rabbit anti-CK19 (Proteintech), Cy3-conjugated donkey anti-rabbit, FITC-conjugated donkey anti-goat, Cy3-conjugated donkey anti-mouse, Cy5-conjugated donkey anti-mouse, and FITC-conjugated donkey anti-rabbit (Jackson Lab).
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