Skin necropsies were snap frozen in Tissue-Tek OCT Compound freezing medium (Sakura Finetek USA Inc.) and HE- and IFS performed on ethanol-fixed tissue sections of 6 µm thickness [9] (link), [13] (link). For detection of MusPV1 L1 protein, sections were stained with a rabbit polyclonal immune serum directed against MusPV1 L1 at a dilution of 1∶4000 and detected with either an Alexa Fluor 488 or an Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (both Life Technologies), as indicated. Co-stainings with either directly conjugated Alexa Fluor 488-anti-mouse CD4 or Alexa Fluor 488-anti-mouse CD8a antibodies (both Biolegend; dilution 1∶100) were performed to detect CD4+ or CD8+ T cells, respectively. To determine localization of MusPV1 L1 in relation to basal keratinocytes, sections were co-stained with a phycoerythrin-conjugated anti-CD49f antibody (integrin alpha 6, BD Biosciences). Nuclei were visualized by mounting sections with ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) (LifeTechnologies). All microscopy analyses were performed on a Zeiss LSM 510 UV system and color levels of images were processed equally in Adobe Photoshop across experiments.
Alexa fluor 594 conjugated donkey anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody is a fluorescently labeled reagent used for the detection and visualization of rabbit primary antibodies in various immunoassays and microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti mouse cd4, by BioLegend (1 mentions)
Prolong gold antifade reagent containing 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
A21207, by Thermo Fisher Scientific (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Rabbit anti brdu, by Merck Group (1 mentions)
Tcs sp8, by Leica (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Primary antibody against nf κb p65, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hcs cell mask deep red, by Thermo Fisher Scientific (1 mentions)
Columbus image analysis software, by PerkinElmer (1 mentions)
Anti p21 antibody, by Abcam (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Anti gfp antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
8 protocols using alexa fluor 594 conjugated donkey anti rabbit secondary antibody
1
Immunostaining of Murine Papillomavirus
2
Immunofluorescence Staining of BV2 Cells
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After been xed with 4% PFA for 30 min at room temperature (RT), BV2 cells were washed with PBS three times and subsequently blocked with PBS containing 0.1% Triton X-100 and 3% BSA for 1 h at RT. Then the cells were incubated with primary antibody of iNOS (18985-1-AP, Proteintech), CD86 (13395-1-AP, Proteintech), CD206 (18704-1-AP, Proteintech) and Arg1 (16001-1-AP, Proteintech) overnight at 4°C. The cells were then washed three times with PBS and incubated with Alexa Fluor 488-conjugated donkey antirabbit secondary antibody (A-21206, Life technologies) or Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (A-21207, Life technologies) for additional 24 h at 4℃. The uorescence images were taken with Leica DMI 8 microscope.
3
LRRK2 Expression in Spinal Cord
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In addition, LRRK2 immunofluorescence was performed in the SC of rats. The staining method was carried out as mentioned above. Primary antibody (ab133476, 1:50 dilution; Abcam) and Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (1:200 dilution; Thermo Fisher Scientific) were used to evaluate local LRRK2 expression in SC in different groups.
4
Quantification of Cumulus Cell Proliferation
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Cumulus cell proliferation was assessed by BrdU incorporation. Mice were injected with 100 mg BrdU/kg body weight 2 h prior to ovary collection. COCs were collected and fixed for 1 h in 4% paraformaldehyde. After 3 washes with PBS containing 0.1% Tween-20, COCs were permeabilized with 0.5% Triton X-100 for 20 min and blocked with 3% BSA in PBS for 30 min, then the COCs were incubated overnight with Rabbit anti-BrdU (100-fold dilution; Sigma) primary antibody. After thorough washing, the COCs were incubated for 1 h with Alexa Fluor 594-conjugated donkey anti-Rabbit secondary antibody (200-fold dilution; Thermo Fisher Scientific). The percentage of BrdU positive cumulus cells in relation to DAPI positive cells was determined by a confocal scanning laser microscopy (Nikon A1R-si) and the NIS Elements Basic Research software.
5
Immunofluorescence analysis of smooth muscle markers
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Cells were fixed with paraformaldehyde and incubated with the following primary antibodies: rabbit polyclonal anti-a-SMA, rabbit polyclonal anti-SM22a, and rabbit polyclonal anti-SM-MHC antibodies for 60 min at room temperature, and then they were washed with PBS for three times. Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (R37119; Thermo Fisher, US) was used to detect the localization of anti-a-SMA, anti-SM22a, and anti-SM-MHC antibodies, respectively. Nuclear staining was done with 4′,6-diamidino-2-phenylindole (DAPI). The images were viewed by a confocal laser scanning microscope (TCS, SP8; Leica, Germany).
6
NF-κB Activation in hNPCs via TNF-α Stimulation
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hNPCs were stimulated with 20 ng/ml TNF-α. After 3 h, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Blocking and permeabilization were carried out in 10% normal donkey serum (NDS; Jackson ImmunoResearch), 3% bovine serum albumin, and 0.3% Triton X-100 (Sigma) prepared in PBS. Samples were incubated at 4 °C overnight with a primary antibody against NF-κB p65 (1:50; Santa Cruz Biotechnology) diluted in PBS containing 3% NDS. After being washed with PBS, slides were labeled with an Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (1:500; Invitrogen, Carlsbad, CA, USA) in PBS containing 3% NDS for 1 h at room temperature. Cells were counterstained using Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (Vector, Burlingame, CA, USA) and visualized by fluorescence microscopy (BX51; Olympus, Center Valley, PA, USA).
7
Immunofluorescent Staining of p21 Protein
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Cells were permeabilised by the addition of 100 µl of 0.1% (v/v) Triton X-100 in PBS per well for 20 min. Thereafter, the cells were blocked by incubation for 1 h with 50 µl blocking buffer (5% (w/v) BSA in PBS) followed by incubation overnight at 4 °C with 50 µl of anti-p21 antibody (Abcam, cat. no. Ab109520) diluted 1:1000 in blocking buffer. The cells were then washed three times for 5 min and incubated with 50 µl of an Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (Invitrogen, cat. no. A21207) diluted 1:500 in blocking buffer for 1 h at room temperature in the dark, after which the cells were washed for 5 min with PBS and stained for 15 min with 50 µl Hoechst (Invitrogen, cat. no. H3570) and HCS CellMask™ Deep Red (Life technologies, cat. no. H32721) diluted 1:10,000 and 1:5000 in PBS, respectively. Finally, the cells were washed twice with PBS and imaged using a Yokogawa CV7000 confocal microscope and the images were analysed using the Columbus Image Analysis software (PerkinElmer).
8
Tracking MSC Homing in Liver Injury
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Mice were transfused with GFP-transgenic mouse MSCs 30 min after ConA injection. After another 7.5 h, livers were harvested. Frozen sections were stained with anti-GFP antibody (Abcam, New Territories, Hong Kong) and labeled with Alexa Fluor 594 conjugated donkey anti-rabbit secondary antibody (Invitrogen) and DAPI (Sigma-Aldrich) to reveal localization of MSCs.
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