The following antibodies were used as primary antibodies for immunofluorescence microscopy: SGO1 (SAB1405371, Sigma Aldrich), GFP (ab290, Abcam) and CENPA (07–574, Millipore; and ab13939, Abcam). For immunoblotting, the following primary antibodies were used: SA1 (ab4457, Abcam), SA2 (A300-158a, Bethyl Laboratories), SMC1 (A300-055A, Bethyl Laboratories), SCC1 (05-908, Millipore), WAPL (A-7, sc-365189, Santa Cruz), Sororin (ab192237, Abcam), HSP90 (sc-13119(F-8), Santa Cruz) and α-tubulin (T5168, Sigma Aldrich). All primary antibodies were used at a 1:1000 dilution with the exception of HSP90 and α-tubulin (1:10000). For coimmunoprecipitation, we used 4.5 μg of SMC1 (A300-055A, Bethyl Laboratories) or IgG (2729 S, Cell Signaling) per sample. Secondary antibodies were used at a 1:1000 dilution. For immunofluorescence microscopy we used: Alexa Fluor 488 goat anti-mouse (A-11001, Life Technology), Alexa Fluor 568 goat anti-mouse (A-11004, Life Technology), Alexa Fluor 488 goat anti-rabbit (A-11008, Life Technology) and Alexa Fluor 568 goat anti-rabbit (A-11011, Life Technology). For western blots, we used the following secondary antibodies: anti-goat-PO (P0449, DAKO), anti-rabbit-PO (P0448, DAKO) and anti-mouse-PO (P0447, DAKO).
Alexa fluor 568 goat anti rabbit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Alexa Fluor 568 goat anti-rabbit is a secondary antibody conjugated with the Alexa Fluor 568 fluorescent dye. It is used to detect and visualize primary rabbit antibodies in various immunoassay techniques such as immunofluorescence, flow cytometry, and Western blotting.
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Lab products found in correlation
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (61 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (23 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (22 mentions)
Alexa fluor 488 goat anti chicken, by Thermo Fisher Scientific (11 mentions)
Alexa fluor 647 goat anti mouse, by Thermo Fisher Scientific (9 mentions)
Hoechst 33342, by Thermo Fisher Scientific (7 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (6 mentions)
Triton x 100, by Merck Group (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Lsm 510, by Zeiss (4 mentions)
Alexa fluor 647 goat anti rabbit, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 568 goat anti rat, by Thermo Fisher Scientific (4 mentions)
Normal goat serum (ngs), by Vector Laboratories (4 mentions)
Goat serum, by Merck Group (4 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Alexa fluor 488 goat anti rat, by Thermo Fisher Scientific (4 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
159 protocols using alexa fluor 568 goat anti rabbit
1
Antibody Characterization for IF and IB
2
Immunofluorescence Staining of Viral Proteins
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For immunofluorescence staining, HRT-18 cells, LA-N-5 cells, and primary murine CNS cell cultures were washed with sterile PBS and then fixed with 4% (wt/vol) paraformaldehyde for 30 min at room temperature. After washing, HRT-18 or LA-N-5 cells were permeabilized with 100% methanol at −20°C for 5 min and then incubated with primary antibody: a monoclonal mouse anti-S protein antibody (1/2 of 4.3.E4 hybridoma supernatant) for 1 h at room temperature. After three washes with PBS, cells were incubated in the dark for 1 h at room temperature with the secondary fluorescent antibody Alexa Fluor 488 goat anti-mouse (1/1,000; Life Technologies). Fixed and permeabilized primary murine cultures were incubated for 1 h at room temperature with a blocking solution (2% bovine serum albumin [BSA] in PBS supplemented with 0.1% Triton X-100) and then stained with primary antibodies: a polyclonal rabbit anti-S protein of bovine coronavirus (BCoV) at a 1/1,000 dilution and mouse monoclonal anti-microtubule-associated protein 2 (MAP2; at a dilution of 1/1,000) for 1 h at room temperature. After three washes with PBS, cells were incubated in the dark for 1 h at room temperature with the secondary fluorescent antibody Alexa Fluor 568 goat anti-rabbit (1/1,000; Life Technologies) or Alexa Fluor 488 anti-mouse (1/1,000; Life Technologies).
3
Immunofluorescent Staining of Keratins
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HeLa and HeLa/SN100 cells were grown on Matek 2.5 cm plates with 1.5 glass coverslips until 80% confluent. Growth media was aspirated off and the cells were washed with PBS. Cells were then fixed with 1 ml of 10% formalin for 20 min at room temperature. Fixed cells were washed three times with PBS and then blocked for 1 h in 1 ml of PBS containing 5% BSA and 0.3% triton X-100. Cells were then incubated with primary antibodies (1:250) anti-Keratin 8 and (1:800) anti-Keratin 18 overnight at 4 °C. Cells were washed three times with PBS before incubation with the secondary antibodies, Alexa Fluor 568 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse (Life Technologies) at 1:1,000 dilutions for 1 h at room temperature. Cells were then washed three times with PBS before imaging using a Nikon TiE inverted widefield fluorescence microscope. Image analysis was performed using ImageJ.
4
Immunofluorescence Imaging of HeyA8 Cells
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Immunostaining was performed as described previously (Chaluvally-Raghavan et al., 2014 (link); Pradeep et al., 2014 (link)). Briefly, HeyA8 cells were grown on eight-well chamber slides (ibidi USA, Madison, WI, USA) then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in blocking solution (0.5% BSA in PBS) followed by blocking for 1 h with 0.5% BSA in PBS, and then stained overnight at 4°C with the indicated primary antibodies. After washing with PBS, cells were incubated with secondary antibodies, Alexa Fluor goat anti-mouse 488 (Cat#38731, Life Technologies, Carlsbad, CA) and Alexa Fluor 568 goat anti-rabbit (Cat#35646, Life Technologies, Carlsbad, CA) for 1 hour at room temperature. Glass slides were mounted using ProLong Gold Antifade Reagent (Life Technologies, Carlsbad, CA) containing DAPI. Images were acquired with a 40X objective using a confocal laser scanning microscope (LSM 510; Zeiss, Oberkochen, Germany) and analyzed using the Aim 4.2 software LSM510.
5
Multicolor Immunofluorescence for Subcellular Imaging
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Cells were fixed in 4% PFA/PBS for 10 min, followed by permeabilisation in 0.2% Triton X‐100/PBS for 15 min, followed by blocking in 2% BSA/PBS for 1 h. Cells were incubated with primary antibodies overnight in a humidified chamber at 4°C followed by extensive washing before addition of secondary antibodies for 1 h at room temperature. Secondary antibodies used for detection were as follows: Alexa Fluor 488 goat anti‐mouse (A11029), Alexa Fluor 488 goat anti‐rabbit (A11034), Alexa Fluor 568 goat anti‐mouse (A11004), Alexa Fluor 568 goat anti‐rabbit (A11011), Alexa Fluor 647 goat anti‐mouse (A21236), Alexa Fluor 647 goat anti‐rabbit (A21245); all purchased from Life Technologies. Slides were mounted in Vectashield antifade aqueous mounting medium (Vector Laboratories), with or without DAPI. For triple colour antibody‐based immunofluorescence, cells were co‐incubated with two antibodies (typically TOM20 and DNA), thoroughly washed and incubated with Alexa Fluor 488/568 secondary antibodies. Following extensive washing, cells were blocked with 5% mouse serum (Life Technologies) for 1 h at room temperature and further incubated with cytochrome c directly conjugated to Alexa Fluor 647 (clone 6H2.B4, Biolegend).
6
Immunohistochemical Analysis of Visual Cortex
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Animals were transcardially perfused with saline and 4% formaldehyde. The brains were removed, cryoprotected in 30% sucrose, and cut into 30 µm coronal sections on a frozen sliding microtome (Physitemp Instruments). Floating sections were blocked for 1 hr at room temperature in Tris buffered saline (TBS) containing 10% normal goat serum and 1% Tween-100, then incubated overnight at 4°C using in the same solution with the following antibodies: chicken anti-GFP, 1:1000 (Aves); rabbit anti-SST, 1:300 (Swant); and rabbit anti-VIP, 1:1000 (Immunostar). The sections were then washed in TBS with 1% Tween-100 three times for 15 min, incubated for 1 hr at room temperature in blocking solution with 1:1000 each of Alexa Fluor 488 goat anti-chicken and Alexa Fluor 568 goat anti-rabbit (Life Technologies). The sections were washed in phosphate buffered saline three times for 10 min, mounted on glass slides, dried, and covered with coverslips. Images of the visual cortex were captured using a Zeiss Axiovert-200 micrscope and AxioCam Mrm (Zeiss). Contrast was adjusted in ImageJ.
7
Immunofluorescence Labeling of Beta-Catenin
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Tissue was fixed in freshly prepared 4% PFA/PBS (EMS) for 1 hr at 4°C, blocked using blocking solution (described above) for 24 hr at 4°C and incubated with rabbit anti-beta-catenin antibody (1:1000 in blocking solution, Sigma PLA0230) for 24 hr at 4°C. Secondary antibody (Alexa Fluor 568 Goat anti-rabbit; Life-Technologies ALL036) was applied at 1:400 in blocking solution. 3 µM DAPI and 130 nM Alexa Fluor-488 Phalloidin (ThermoFisher) were used as counterstains. 12 × 20 min PBST washes were performed following both primary and secondary antibodies.
8
Cytoskeletal dynamics and cell-cell adhesion
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Actin filaments were stained with Alexa Fluor 568 Phalloidin (Life Technologies). Myosin IIA was stained with either Rabbit Anti-Myosin IIA (Sigma M8064) or Mouse anti Myosin IIA (abcam ab55456). Paxillin was stained with Rabbit Monoclonal anti-Paxillin [Y113] (abcam ab32084). Cofilin was stained with Rabbit anti cofilin (abcam ab11062). E-cadherin was stained with Rat anti E-cadherin (Sigma U3254). Secondary antibodies used were Alexa Fluor 488 goat anti rabbit (Life Technologies A11011), Alexa Fluor 568 Goat anti Rabbit (Life Technologies A11008), Alexa Fluor 488 Goat anti Mouse (Life Technologies A11001), Alexa Fluor 568 Goat anti-Mouse (Life Technologies A11004), Alexa Fluor 488 Donkey anti Rat (Life Technologies A21208).
Cells were fixed with either methanol at -20°C for cofilin or with 4% PFA at 37°C for others, permeablized with 0.2% Triton X-100 in TBS, blocked with 1% BSA in TBS and stained with the aforementioned antibodies. Quantitative imaging was performed on a Nikon A1R scanning confocal microscope with either a 1.27 N.A. 60X water immersion objective or a 1.49 N.A. 100X oil immersion objective. For E-cadherin staining, images were taken with an Olympus spinning disk confocal microscope with a super-resolution module installed, 1.49 N.A. 100X objective.
Cells were fixed with either methanol at -20°C for cofilin or with 4% PFA at 37°C for others, permeablized with 0.2% Triton X-100 in TBS, blocked with 1% BSA in TBS and stained with the aforementioned antibodies. Quantitative imaging was performed on a Nikon A1R scanning confocal microscope with either a 1.27 N.A. 60X water immersion objective or a 1.49 N.A. 100X oil immersion objective. For E-cadherin staining, images were taken with an Olympus spinning disk confocal microscope with a super-resolution module installed, 1.49 N.A. 100X objective.
9
Immunofluorescent Labeling of Brain Tissue
10
Immunofluorescence Quantification of E/N-Cadherin
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Relative expression levels of E cadherin and N cadherin were assessed via immunofluorescence staining. Cells were fixed with 4 % paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA), and stained with mouse monoclonal antibodies against human E-cadherin and rabbit polyclonal antibodies against human N-cadherin (Abcam, USA). Cells were subsequently incubated with appropriate secondary antibodies: Alexa Fluor-488 goat anti-mouse and Alexa Fluor-568 goat anti-rabbit (Life Technologies, USA). Stained cells were examined under a fluorescence microscope (Zeiss, LSM 800 Confocal, USA). Fluorescence intensity was measured with Zen 2.1 software (Zeiss, Blue edition, USA), with n=4. Intensity data were transformed into graphs, and analyzed using GraphPad Prism 9 (GraphPad Holdings, USA).
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