Three coronal sections from each mouse (450-μm interval) were incubated with 10% normal donkey serum for 1 hour and then with the following primary antibodies for overnight at 4℃: rabbit polyclonal anti-doublecortin (anti-DCX, 1:1,000; Millipore Corp., Burlington, MA, USA), mouse monoclonal anti-human nuclei (hNU, 1:200; Millipore Corp.). After washing with PBS, the sections were incubated with the following secondary antibodies for 2 hours at room temperature (22℃±3℃): Alexa488-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA), Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch). Then, after washing with PBS, the sections were mounted with DAPI (4, 6-diamidino-2-phenylindole)-included mounting media and observed using a confocal microscopy (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany).
Alexa488 conjugated donkey anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa488-conjugated donkey anti-mouse IgG is a secondary antibody used to detect and visualize mouse immunoglobulin G (IgG) in various immunoassays. The antibody is conjugated with the Alexa Fluor 488 fluorescent dye, which emits green fluorescence upon excitation, allowing for the detection and quantification of target mouse IgG molecules.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (2 mentions)
Anti dcx, by Merck Group (1 mentions)
Cy3 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Mouse anti α sma, by Merck Group (1 mentions)
Mouse anti vimentin antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa 594 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Igg free bsa, by Merck Group (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Dfc7000t, by Leica Microsystems (1 mentions)
M205 fca, by Leica Microsystems (1 mentions)
3 protocols using alexa488 conjugated donkey anti mouse igg
1
Immunohistochemical Analysis of Neurogenesis
2
Double Immunofluorescence Staining of Kidney
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For double staining of vimentin with collagen type IV or α‐SMA with collagen type IV, immunofluorescence staining was performed on 4‐µm‐thick sections as described previously (Hamatani et al., 2018 ). Kidney sections were deparaffinized and rehydrated, then antigen retrieval was performed by microwave heating in 10 mM citric acid buffer, pH 6.0 for α‐SMA, and pH 7.0 for vimentin. Endogenous biotin activity was blocked with an Avidin/biotin blocking kit (Vector Laboratories), and nonspecific binding was blocked with a background buster (Accurate Chemical & Scientific, Westbury, NY). Antibodies were diluted in 1% IgG‐free BSA (Sigma‐Aldrich) in PBS. Mouse anti–α‐SMA (Sigma‐Aldrich) or mouse anti‐vimentin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated overnight at 4°C, followed by Alexa 488‐conjugated donkey anti‐mouse IgG (Jackson ImmunoResearch) for 1 hr at room temperature. We have reported that these antibodies colocalized with the PEC marker PAX2 (Naito, Pippin, & Shankland, 2014 ). Biotinylated goat anti‐collagen type IV (Southern Biotech, Birmingham, AL) was incubated overnight at 4°C, followed by Alexa 594‐conjugated streptavidin (Life Technologies) for 1 hr at room temperature. The sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories).
3
Cryopreservation and Transplantation of Primordial Germ Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A male PGC line (W4) was used in the gonadal migration assay. Cultured PGCs were fluorescently labeled with PKH-26 (Zynaxis, Malvern, PA, USA), as described by Yamamoto et
al. (2007) [21 (link)]. A few labeled PGCs were later used for transplantation. The remaining labeled PGCs were immediately suspended in either DTS
or PTS cryomedium and then cryopreserved in aliquots of approximately 1 × 105 cells per vial at −196°C for > 1 month. Approximately 1 × 103 unfrozen or frozen-thawed
PGCs were intravascularly injected into the dorsal aorta of BPR embryos at stages 15–16 through a window (~20 mm diameter) in the narrow end of the eggshell. After sealing the window in the
eggshell with cling film, the manipulated embryos were incubated for six days in total, until they reached stage 29. Whole gonads attached to the adjacent mesonephros were collected.
Immunofluorescence of whole-mount gonads was carried out as previously reported [22 ], using an anti-chicken vasa homolog (CVH) monoclonal antibody
(1:5; [23 ]) followed by Alexa 488-conjugated donkey anti-mouse IgG (1:400; Jackson ImmunoResearch, West Grove, PA, USA). The number of CVH+PKH-26-labeled cells observed in both the left and right gonads was counted under a fluorescence microscope (M205FCA; Leica Microsystems, Wetzlar, Germany) and photographed using a CCD
camera (DFC7000T; Leica Microsystems).
al. (2007) [21 (link)]. A few labeled PGCs were later used for transplantation. The remaining labeled PGCs were immediately suspended in either DTS
or PTS cryomedium and then cryopreserved in aliquots of approximately 1 × 105 cells per vial at −196°C for > 1 month. Approximately 1 × 103 unfrozen or frozen-thawed
PGCs were intravascularly injected into the dorsal aorta of BPR embryos at stages 15–16 through a window (~20 mm diameter) in the narrow end of the eggshell. After sealing the window in the
eggshell with cling film, the manipulated embryos were incubated for six days in total, until they reached stage 29. Whole gonads attached to the adjacent mesonephros were collected.
Immunofluorescence of whole-mount gonads was carried out as previously reported [22 ], using an anti-chicken vasa homolog (CVH) monoclonal antibody
(1:5; [23 ]) followed by Alexa 488-conjugated donkey anti-mouse IgG (1:400; Jackson ImmunoResearch, West Grove, PA, USA). The number of CVH+PKH-26-labeled cells observed in both the left and right gonads was counted under a fluorescence microscope (M205FCA; Leica Microsystems, Wetzlar, Germany) and photographed using a CCD
camera (DFC7000T; Leica Microsystems).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!