Mice were anesthetized with urethane (1.3 g/kg) via intraperitoneal injection and perfused transcardially through the left ventricle with saline, followed by 0.1 M phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde. The brain was dissected and fixed overnight in the same fixation solution at 4 °C and stored in 30% sucrose in 0.05 M PB. To evaluate the necdin expression in the CNS, serial coronal sections were cut into 50-μm-thick sections using a freezing microtome. Slices were incubated for 1 h at room temperature in phosphate-buffered saline containing 0.03% Triton X-100 (PBST), 2% bovine serum albumin, and 10% normal goat serum and then incubated overnight at 4 °C in PBST containing 1/1000 dilution of rabbit antibodies against necdin (Bio Academia, Osaka, Japan) for 40 h at 4 °C. After rinsing with PBST, tissue slices were incubated with the secondary antibodies for 3 h: 1/200 dilution of goat anti-rabbit IgG-conjugated Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, PA, USA). After rinsing with PB, the slices were mounted with RapiClear 1.47 (SunJin Lab, Hsinchu City, Taiwan), and a cover slip was placed. Immunofluorescence images were observed using a Leica SP8 confocal microscope (Leica microsystems, Wetzlar, Germany).
Alexa fluor 594 conjugated goat anti rabbit igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor 594-conjugated goat anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. It is labeled with the Alexa Fluor 594 fluorescent dye, which can be detected using a red fluorescence detection system.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 confocal microscope, by Leica (4 mentions)
Alexa fluor 488 conjugated sheep anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (2 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions)
Rhodamine phalloidin, by Cytoskeleton (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti ly6c hk1, by BioLegend (1 mentions)
Tritc conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Anti cd31 mec13.3, by Thermo Fisher Scientific (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Anti cd45, by Thermo Fisher Scientific (1 mentions)
Zen 2010, by Zeiss (1 mentions)
Supermount, by BioGenex (1 mentions)
C1 confocal imaging system, by Nikon (1 mentions)
Glial fibrillary acidic protein (gfap), by Proteintech (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Triton, by Merck Group (1 mentions)
Cryobloc compound, by Diapath (1 mentions)
Axio imager m2p, by Zeiss (1 mentions)
14 protocols using alexa fluor 594 conjugated goat anti rabbit igg
1
Necdin Expression in Mouse CNS
2
Necdin Expression Evaluation in CNS
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Mice were anesthetized with urethane (1.3 g/kg) via intraperitoneal injection and perfused transcardially through the left ventricle with saline, followed by 0.1 M phosphate-buffer (PB, pH 7.4) containing 4% paraformaldehyde. The brain was dissected and xed overnight in the same xation solution at 4°C and stored in 30% sucrose in 0.05 M PB. To evaluate the necdin expression in the CNS, serial coronal sections were cut into 50-μm-thick sections using a freezing microtome. Slices were incubated for 1 h at room temperature in phosphate-buffered saline containing 0.03% Triton X-100 (PBST), 2% bovine serum albumin, and 10% normal goat serum and then incubated overnight at 4°C in PBST containing 1/1000 dilution of rabbit antibodies against necdin (Bio Academia, Osaka, Japan) for 40 h at 4°C. After rinsing with PBST, tissue slices were incubated with the secondary antibodies for 3 h: 1/200 dilution of goat antirabbit IgG-conjugated Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, PA, USA). After rinsing with PB, the slices were mounted with RapiClear 1.47 (SunJin Lab, Hsinchu City, Taiwan), and a cover slip was placed. Immuno uorescence images were observed using a Leica SP8 confocal microscope (Leica microsystems, Wetzlar, Germany).
3
Necdin Expression in the CNS
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Mice were anesthetized with urethane (1.3 g/kg) via intraperitoneal injection and perfused transcardially through the left ventricle with saline, followed by 0.1 M phosphate-buffer (PB, pH 7.4) containing 4% paraformaldehyde. The brain was dissected and xed overnight in the same xation solution at 4 °C and stored in 30% sucrose in 0.05 M PB. To evaluate the necdin expression in the CNS, serial coronal sections were cut into 50-µm-thick sections using a freezing microtome. Slices were incubated for 1 h at room temperature in phosphate-buffered saline containing 0.03% Triton X-100 (PBST), 2% bovine serum albumin, and 10% normal goat serum and then incubated overnight at 4 °C in PBST containing 1/1000 dilution of rabbit antibodies against necdin (Bio Academia, Osaka, Japan) for 40 h at 4 °C. After rinsing with PBST, tissue slices were incubated with the secondary antibodies for 3 h: 1/200 dilution of goat antirabbit IgG-conjugated Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, PA, USA). After rinsing with PB, the slices were mounted with RapiClear 1.47 (SunJin Lab, Hsinchu City, Taiwan), and a cover slip was placed. Immuno uorescence images were observed using a Leica SP8 confocal microscope (Leica microsystems, Wetzlar, Germany).
4
Necdin Expression in Mouse CNS
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Mice were anesthetized with urethane (1.3 g/kg) via intraperitoneal injection and perfused transcardially through the left ventricle with saline, followed by 0.1 M phosphate-buffer (PB, pH 7.4) containing 4% paraformaldehyde.
The brain was dissected and fixed overnight in the same fixation solution at 4°C and stored in 30% sucrose in 0.05 M PB. To evaluate the necdin expression in the CNS, serial coronal sections were cut into 50-μm-thick sections using a freezing microtome. Slices were incubated for 1 h at room temperature in phosphate-buffered saline containing 0.03% Triton X-100 (PBST), 2% bovine serum albumin, and 10% normal goat serum and then incubated overnight at 4°C in PBST containing 1/1000 dilution of rabbit antibodies against necdin (Bio Academia, Osaka, Japan) for 40 h at 4°C. After rinsing with PBST, tissue slices were incubated with the secondary antibodies for 3 h: 1/200 dilution of goat anti-rabbit IgG-conjugated Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, PA, USA). After rinsing with PB, the slices were mounted with RapiClear 1.47 (SunJin Lab, Hsinchu City, Taiwan), and a cover slip was placed. Immunofluorescence images were observed using a Leica SP8 confocal microscope (Leica microsystems, Wetzlar, Germany).
The brain was dissected and fixed overnight in the same fixation solution at 4°C and stored in 30% sucrose in 0.05 M PB. To evaluate the necdin expression in the CNS, serial coronal sections were cut into 50-μm-thick sections using a freezing microtome. Slices were incubated for 1 h at room temperature in phosphate-buffered saline containing 0.03% Triton X-100 (PBST), 2% bovine serum albumin, and 10% normal goat serum and then incubated overnight at 4°C in PBST containing 1/1000 dilution of rabbit antibodies against necdin (Bio Academia, Osaka, Japan) for 40 h at 4°C. After rinsing with PBST, tissue slices were incubated with the secondary antibodies for 3 h: 1/200 dilution of goat anti-rabbit IgG-conjugated Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, PA, USA). After rinsing with PB, the slices were mounted with RapiClear 1.47 (SunJin Lab, Hsinchu City, Taiwan), and a cover slip was placed. Immunofluorescence images were observed using a Leica SP8 confocal microscope (Leica microsystems, Wetzlar, Germany).
5
Visualizing Apicomplexan Protein Localization
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For Immunofluorescence assays (IFA), infected confluent VERO monolayers were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and blocked with 1% bovine serum albumin in PBS (BSA-PBS). The primary antibodies used were mouse anti-TgRuvBL1 (1:500), mouse anti-TgRNase H1 (1:500), mouse anti-TgRPS2 (1:500), and rabbit anti-SAG1 (1:4,000). The secondary antibodies used were Alexa Fluor 488-conjugated goat anti-mouse IgG (1:2,000; Thermo Fisher Scientific Inc.) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:2,000; Jackson ImmunoResearch, West Grove, PA, United States). Images were acquired with a LSM880 confocal laser scanning microscope (Zeiss, Germany) after the samples were stained with DAPI.
6
Immunofluorescence Imaging of RASD1 Protein
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Cells were grown on 13-mm-diameter coverslips for 2 days and then fixed with 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. After being blocked in 10% normal goat serum for 30 min, the cells were incubated with rabbit anti-RASD1 antibody (Abcam, 1:200) in a humidified chamber overnight at 4 °C. On the following day, the Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200, Jackson lab) was added and incubated for 2–3 h at room temperature. F-actin was stained using 200 μL of 100 nM rhodamine-phalloidin according to the manufacturer’s protocol (Cytoskeleton, Denver, CO). The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; 1:1000; Sigma). The cells were observed and recorded using a fluorescence microscope.
7
Immunofluorescence Staining of Thymus Tissue
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Thymus tissues were embedded in OCT compound and snap-frozen in liquid nitrogen. Ten-micrometre-thick cryosections were air-dried and fixed for 10 min in cold acetone. The cryosections were blocked for 1 h in PBS containing 2% FBS and 1 mg ml−1 anti-FcγRII/CD16 (2.4G2; in-house production). The cryosections were incubated overnight at 4 °C with the following antibodies at 2.5 μg ml−1 working concentration: anti-CD31 (MEC13.3; eBioscience), anti-Ly6C (HK1.4; BioLegend) and anti-CD45.2 (104; eBioscience). Anti-collagen IV (LSL-LB-1407; Cosmo Bio Co., LTD) was diluted 1:1,000. Unconjugated antibodies were detected with the following secondary antibodies: AlexaFluor594-conjugated goat anti-rabbit IgG (Jackson, ZF-0516) and TRITC-conjugated streptavidin (Jackson, 016-020-084). The detailed information of these antibodies is also listed in Supplementary Table 4 . Microscopical analysis was performed using a confocal microscope (Zeiss LSM-710) and the images were processed with ZEN 2010 software (Carl Zeiss, Inc.).
8
Detecting Aggresome-like Inclusions in N27 Cells
9
Immunofluorescence Labeling of Brain Markers
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Immunofluorescence labeling was performed using previously established procedures.18 , 19 The frozen sections were incubated with a mixture of the following primary antibodies overnight at 4°C: STK24 (rabbit, polyclonal; 1:50, bs‐7599R; BIOSS, China), MAP‐2 (guinea pig, polyclonal; 1:200, 188004; SYSY, Germany), and GFAP (mouse, monoclonal; 1:100, 60190‐1‐Ig; Proteintech, China).
On the next day, the sections were incubated with a mixture of corresponding secondary antibodies at room temperature for 1 hour: Alexa Fluor 488‐conjugated donkey anti‐mouse IgG (1:50; 115‐545‐003; Jackson ImmunoResearch, USA), Alexa Fluor 405‐conjugated donkey anti‐guinea pig IgG (1:50; 106‐475‐003; Jackson ImmunoResearch, USA), and Alexa Fluor 594‐conjugated goat anti‐rabbit IgG (1:50; 111‐585‐003; Jackson ImmunoResearch, USA) shielded from light for 60 minutes at 37°C. Images were captured by a confocal laser scanning microscope (A1 + R, Nikon, Japan).
On the next day, the sections were incubated with a mixture of corresponding secondary antibodies at room temperature for 1 hour: Alexa Fluor 488‐conjugated donkey anti‐mouse IgG (1:50; 115‐545‐003; Jackson ImmunoResearch, USA), Alexa Fluor 405‐conjugated donkey anti‐guinea pig IgG (1:50; 106‐475‐003; Jackson ImmunoResearch, USA), and Alexa Fluor 594‐conjugated goat anti‐rabbit IgG (1:50; 111‐585‐003; Jackson ImmunoResearch, USA) shielded from light for 60 minutes at 37°C. Images were captured by a confocal laser scanning microscope (A1 + R, Nikon, Japan).
10
Immunofluorescence Analysis of Xenograft Tumors
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Frozen sections (10 μm) in Cryobloc Compound (Diapath, Martinengo, Italy) of tumour explanted from xenograft tumour‐bearing mice, treated or not with the NAAA inhibitor AM9053 (20 mg·kg−1, peritumourally), were fixed in 4% paraformaldehyde and then blocked with PBS containing 2% BSA, 2% goat serum and 0.1% Triton (all from Sigma‐Aldrich, Milan, Italy) for 60 min at room temperature and incubated overnight at 4°C with the human anti‐Ki67 (1:400, Abcam, Cambridge, UK). All sections were then incubated for 30 min at room temperature with Alexa Fluor 594‐conjugated goat anti‐rabbit IgG (1:1000; Jackson ImmunoResearch, Cambridge, UK), followed by incubation for 5 min at room temperature with DAPI (1:25,000; Invitrogen, Waltham, MA, USA). Sections were mounted with glycerol (Sigma‐Aldrich, Milan Italy) and acquired/analysed by using Axio Imager M2P combined with high‐end fluorescence imaging system ApoTome.2 (Zeiss, Jena, Germany). Images were acquired with an objective 20×.
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