Mice were overdosed with isoflurane and perfused transcardially with (4°C) phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were extracted and stored overnight in PFA at 4°C and transferred to PBS solution the following day. Brains were sliced into 50 µm coronal sections with a vibratome (Leica, VT100S) and collected in cold PBS. Sections were blocked for 2 h at room temperature in 1× phosphate-buffered saline + 2% Triton (PBS-T) and 5% normal goat serum (NGS) on a shaker. Sections were transferred to well plates containing primary antibodies made in PBS-T (1:1000 rabbit anti-c-Fos [SySy]; 1:5000 chicken anti-GFP [Invitrogen]) and incubated on a shaker at 4°C for 48 h. Sections were then washed in PBS-T for 10 min (×3), followed by a 2 h incubation with secondary antibody (1:200 Alexa 555 anti-rabbit [Invitrogen]; 1:200 Alexa 488 anti-chicken [Invitrogen] made in PBS-T). Following three additional 10 min washes in PBS-T, sections were mounted onto micro slides (VWR International, LCC). Nuclei were counterstained with DAPI added to Vectashield HardSet Mounting Medium (Vector Laboratories, Inc), slides were then coverslipped and put in the fridge overnight. The following day the edges were sealed with clear nail-polish and the slides were stored in a slide box in the fridge until imaging.
Alexa 488 anti chicken
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488 anti-chicken is a fluorescently labeled antibody that specifically binds to chicken proteins. It can be used in various immunological techniques, such as flow cytometry and immunofluorescence microscopy, to detect and quantify chicken proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Chicken anti gfp, by Thermo Fisher Scientific (7 mentions)
Lsm 510, by Zeiss (4 mentions)
Lsm 700 confocal microscope, by Zeiss (4 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Alexa 568 anti mouse, by Thermo Fisher Scientific (2 mentions)
Micro slides, by Avantor (2 mentions)
Vectashield hart set mounting medium with dapi, by Vector Laboratories (2 mentions)
Rat anti flag, by Novus Biologicals (2 mentions)
Ss8x9 secureseal spacers, by Thermo Fisher Scientific (2 mentions)
Rabbit anti th, by Merck Group (2 mentions)
Rat anti cd8, by Thermo Fisher Scientific (2 mentions)
Alexa 647 anti mouse, by Thermo Fisher Scientific (2 mentions)
Alexa 488 anti rat, by Thermo Fisher Scientific (2 mentions)
Slowfade gold, by Thermo Fisher Scientific (2 mentions)
Rabbit anti ha, by Cell Signaling Technology (2 mentions)
Anti mouse cy3, by Jackson ImmunoResearch (2 mentions)
Mouse anti nc82, by Developmental Studies Hybridoma Bank (2 mentions)
Vt100, by Leica (1 mentions)
Vectashield hardset mounting medium, by Vector Laboratories (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
10 protocols using alexa 488 anti chicken
1
Immunohistochemistry of Brain Tissue
2
BrdU Labeling of Sox14 Expressing Cells
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Wild-type C57BL/6 dams were mated with heterozygous Sox14Gfp/+ males. BrdU (Sigma, B5002) was administered intraperitoneally to pregnant mice (0.2 mg g−1) at 09:00 between E10.5 and 13.5, estimated from the occurrence of a vaginal plug (morning of the day the plug was detected was designated E0.5). Gfp heterozygous pups were perfused at P7 with 4% paraformaldehyde (PFA) and treated for cryosectioning as above. For BrdU/Sox14 double labelling, 40 μm floating sections were cut, followed by denaturation with 1 M HCl in H2O at 45 °C for 30 min and neutralization with three washes of phosphate-buffered saline (PBS; pH 7.4) for 10 min. Sections were then blocked in 2% normal goat serum (NGS), 0.3% Triton-X, in PBS for 1 h at room temperature, and probed with rat antiBrdU (1:200, in 2% NGS, 0.3% Triton-X, in PBS, OBT0030CX Bio-Rad) and chicken anti-GFP (Ab13970 Abcam) primary antibodies at 4 °C for 48 h, followed by fluorescent Goat Alexa-568 anti-rat and Alexa-488 anti-chicken (A11039, Invitrogen) secondary antibodies. Counting of BrdU+ and Gfp+ double-positive cells in the Sox14Gfp/+ dLGN used every second section, covering the whole span of the dLGN (n=3 brains per developmental stage).
3
Confocal Imaging of Neuronal Markers
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Immunohistochemistry was done as described91 (link) except that the blocking step was overnight at 4°C. Primary antibodies: mouse anti-nc82 (DSHB, AB_2314866) 1:40, chicken anti-GFP (Abcam, ab13970) 1:1000, mouse anti-ChAT4B (DSHB, AB_528122), rabbit anti-GABA (Sigma, A2052). Secondary antibodies: Alexa-568 anti-mouse (Invitrogen) 1:400, Alexa-488 anti-chicken (Invitrogen) 1:400, Alexa-633 anti-mouse (Invitrogen) 1:400, Alexa-568 anti-rabbit (Invitrogen) 1:400.
Prolonged incubation (2-3 days at 4°C) with primary and secondary antibodies was required for homogeneous staining. Specimens were whole mounted in Vectashield (Vector Labs) on charged slides to avoid movement. Confocal stacks were acquired using a Zeiss 780 confocal microscope. Brains were imaged at 768 x 768 pixel resolution every 1 μm (0.46 x 0.46 x 1 μm) using an EC Plan-Neofluar 40x/1.30 oil objective and 0.6 zoom factor. All images were acquired at 16-bit color depth. Maximum projections of z stacks were made in Fiji.89 (link)
Prolonged incubation (2-3 days at 4°C) with primary and secondary antibodies was required for homogeneous staining. Specimens were whole mounted in Vectashield (Vector Labs) on charged slides to avoid movement. Confocal stacks were acquired using a Zeiss 780 confocal microscope. Brains were imaged at 768 x 768 pixel resolution every 1 μm (0.46 x 0.46 x 1 μm) using an EC Plan-Neofluar 40x/1.30 oil objective and 0.6 zoom factor. All images were acquired at 16-bit color depth. Maximum projections of z stacks were made in Fiji.89 (link)
4
Immunohistochemical Analysis of Brain Samples
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Immunohistochemistry follows protocols previously reported15 (link),16 ,18 (link). Mice were overdosed with 3% isoflurane and perfused transcardially with cold (4 °C) phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were extracted and stored overnight in PFA at 4 °C. Fifty μm coronal sections were collected in serial order using a vibratome and collected in cold PBS. Sections were blocked for 1 hour at room temperature in PBST and 5% normal goat serum (NGS) or Bovine albumin serum (BSA) on a shaker. Sections were transferred to wells containing primary antibodies (1:1000 guinea anti-c-Fos [SySy]; 1:1000 rabbit anti-RFP [Rockland]; 1:5000 chicken anti-GFP [Invitrogen]) and allowed to incubate on a shaker overnight at room temperature or 3 days at 4 degrees C. Sections were then washed in PBST for 10-min (x3), followed by 2-hour incubation with secondary antibody (1:200 Alexa 555 anti-rabbit [Invitrogen]; 1:200 Alexa anti-guinea 647 [Invitrogen]; 1:200 Alexa 488 anti-chicken [Invitrogen]). Following three additional 10-min washes in PBST, sections were mounted onto micro slides (VWR International, LLC). Vectashield Hart Set Mounting Medium with DAPI (Vector Laboratories, Inc) was applied, slides were coverslipped, and allowed to dry overnight.
5
Quantification of Neurogenesis in the Dentate Gyrus
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For triple-labelling, free-floating 1-in-12 section series were rinsed and incubated as described above except for H2O2 pre-treatment but 1 h PBS+ blocking instead. Anti-BrdU (rat, 1∶500, AbD Serotex) anti-GFP (chicken, 1∶250, Novus Biologicals) and anti-NeuN (mouse, 1∶100, Millipore) were used as primary antibodies. BrdU denotes newly generated cells, while BrdU/Nestin (Nestin/GFP) and BrdU/NeuN denote new neural precursor cells and new neurons, respectively. After incubation for 48 h at 4°C, RhodamineX (anti-rat, 1∶250, dianova), Alexa 488 (anti-chicken, 1∶1000, Invitrogen) and Alexa 647 (anti-mouse, 1∶300, dianova.) as secondary antibodies diluted in PBS+ were applied for 4 h at RT. Sections were mounted on microscope slides and coverslipped for later quantification.
For stereological counting of cells in the GCL of the DG including the cells in the SGZ in order to determine its total cell number, separate 1-in-12 series of sections were incubated with the fluorochrome 4′,6-diamidino-2-phenylindole (DAPI), which binds to the DNA thereby labeling cell nuclei in general. For this purpose, sections were incubated with PBS-diluted DAPI (1∶1000, Thermo Scientific) for 7 min and afterwards mounted on microscope slides and coverslipped for later quantification.
For stereological counting of cells in the GCL of the DG including the cells in the SGZ in order to determine its total cell number, separate 1-in-12 series of sections were incubated with the fluorochrome 4′,6-diamidino-2-phenylindole (DAPI), which binds to the DNA thereby labeling cell nuclei in general. For this purpose, sections were incubated with PBS-diluted DAPI (1∶1000, Thermo Scientific) for 7 min and afterwards mounted on microscope slides and coverslipped for later quantification.
6
Whole-Mount Brain Immunostaining Protocol
7
Immunohistochemistry for c-Fos and GFP
8
Immunohistochemical Analysis of Brain Sections
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Mice were overdosed with 3% isoflurane and perfused transcardially with cold (4 °C) phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were extracted and kept in PFA at 4 °C for 24 to 48 h and then transferred to PBS solution. Brains were sectioned into 50-μm-thick coronal sections with a vibratome and collected in cold PBS. Sections were blocked for 1 to 2 h at room temperature in PBS combined with 0.2% Triton (PBST) and 5% normal goat serum (NGS) on a shaker. Sections were incubated in primary antibody (1:1,000 rabbit anti-cFos [SySy]; 1:5,000 chicken anti-GFP [Invitrogen]) made in PBST/NGS at 4 °C for 48 h. Sections then underwent three washes in PBST for 10 min each, followed by a 2-h incubation period at room temperature with secondary antibody (1:200 Alexa 555 anti-rabbit [Invitrogen]; 1:200 Alexa 488 anti-chicken [Invitrogen]) made in PBST/NGS. Sections then underwent three more wash cycles in PBST. Sections were mounted onto microscope slides (VWR International). Vectashield HardSet Mounting Medium with DAPI (Vector Laboratories) was applied and slides were coverslipped and allowed to dry overnight. Once dry, slides were sealed with clear nail polish on each edge and stored in a slide box in the refrigerator. If not mounted immediately, sections were stored in PBS at 4 °C.
9
Whole-Mount Immunostaining of Fly Brains
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Immunostaining of whole-mount brains or VNCs was performed as follows: brains or VNCs of 3- to 7-day-old flies were dissected in PBS, fixed in 4% paraformaldehyde (PFA) for 20 min, and subjected to chicken anti-GFP (1:1000, In-vitrogen), rabbit anti-TH (1:1000, Millipore), rat anti-CD8 (1:200, Invitrogen), mouse anti-nc82 (1:25, Developmental Studies Hybridoma Bank), or all four at 4°C for 18–40 hr, followed by incubation with fluorescent Alexa 488 anti-chicken (1:1000, Invitrogen), Alexa 488 anti-rat (1:200, Invitrogen), Alexa 568 anti-rabbit (1:1000, Invitrogen), Alexa 647 anti-mouse (1:1000, Invitrogen), or Cy3 anti-mouse (1:200, Jackson Immunoresearch) secondary antibodies for ~16 hr at 4°C. Brains or VNCs were mounted in Vectashield (Vector Laboratories) or SlowFade Gold using SS8X9-SecureSeal spacers (both Thermo Fisher Scientific). Images were obtained on an LSM510 or LSM700 confocal microscope (Zeiss) with 1-μm- or 3-μm-thick sections under 10x or 25x magnification. MultiColor FlpOut (MCFO) analysis of select driver combinations was performed as described previously (Nern et al., 2015 (link)) using rat anti-FLAG (1:200, Novus Biologicals) and rabbit anti-HA (1:300, Cell Signaling Technology) antibodies.
10
Whole-Mount Immunostaining of Fly Brains
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