Chicken anti gfp, by Rockland Immunochemicals - Product details

1

Unilateral Antennal Ablation and Fly Brain Dissection

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Adults were aged at 20 °C for 5–7 days before performing antennal ablation (Paglione et al., 2020 (link)). Unilateral antennal ablation (e.g., removal of one antenna) was performed using high precision and ultra-fine tweezers, and flies returned to vial for the appropriate time. The ablation of 3^rd antennal segments did not damage the rest of the head or lead to fly mortality. At corresponding time points, adult brain dissections were performed as described (Paglione et al., 2020 (link)): decapitated heads were fixed in 4% formaldehyde in PTX (0.5% Triton X-100 in PBS) for 20 min, and washed 3x10 min with PTX. Brain dissections were performed in PTX, and dissected brains were fixed in 4% formaldehyde in PTX for 10 min, followed by 1 hr of blocking in 10% normal goat serum (Jackson Immuno) in PTX and an O/N incubation with the following primary antibodies at 4 °C in blocking solution: 1:500 chicken anti-GFP (Rockland), and 1:150 mouse anti-nc82 (DSHB, nc82). Brains were then washed 3x10 min with PTX at RT, and incubated with secondary antibodies in PTX at RT for 2 hr: 1:200 Dylight 488 goat anti-chicken (abcam, ab96947), and 1:200 AlexaFluor 546 goat anti-mouse (ThermoFisher, a-11030). Brains were washed 3x10 min with PTX at RT, and mounted in Vectashield for microscopy.

Llobet Rosell A., Paglione M., Gilley J., Kocia M., Perillo G., Gasparrini M., Cialabrini L., Raffaelli N., Angeletti C., Orsomando G., Wu P.H., Coleman M.P., Loreto A, & Neukomm L.J. (2022). The NAD+ precursor NMN activates dSarm to trigger axon degeneration in Drosophila. eLife, 11, e80245.

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Antibodies Used for Neuronal Immunostaining

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Antibodies used for immunostaining included: mouse anti-nc82 (1:50, Developmental Studies Hybridoma Bank), rat-anti-Chinmo (1:500; Wu et al., 2012 (link)), rabbit anti-Br-Z3 (1:250; this study), rabbit anti-GFP (1:1000; Invitrogen), chicken anti-GFP (1:500; Rockland Immunochemicals), rabbit anti-dsRed (1:500; Clontech), mouse anti-mCherry (1:500; Clontech), rabbit anti-β-gal (1:500; MP Biomedicals), chicken anti-β-gal (1:500; Abcam). Most primary antibodies were preabsorbed against fixed embryos. Secondary goat antibodies were conjugated to Alexa Fluor 488, 568 or 633 (Molecular Probes). Slides were mounted in Vectashield (Vector Labs) or dehydrated through an ethanol series and mounted in DPX. Images were collected on a Leica SP5 (Light Microscopy Imaging Center, Indiana University). Confocal stacks were merged using Leica LSM software. Samples that were directly compared were prepared under identical conditions and imaged in parallel.

Wu Y.C., Chawla G, & Sokol N. (2020). let-7-Complex MicroRNAs Regulate Broad-Z3, Which Together with Chinmo Maintains Adult Lineage Neurons in an Immature State. G3: Genes|Genomes|Genetics, 10(4), 1393-1401.

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Immunohistochemical Labeling of Barrel Cortex

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Barrel cortex sections were washed in TRIS buffer (TB) for 15 min, TRIS-buffered saline (TBS) for 15 min and TBS + 0.5% Triton X-100 (TBST) for 2×15 min, all at pH 7.6. Blocking was done for 90 min at room temperature in 0.25% bovine serum albumin/10% goat serum/TBST (Jackson Immuno Research). Sections were incubated for 48–72 h at 4°C with primary antibodies (i) chicken anti-GFP (Aves) diluted 1:1000, (ii) mouse anti-RFP (Rockland) diluted 1:1000, and (iii) rabbit anti-PV (Swant) diluted 1:5000 in blocking solution. After washing 4×15 min with TBST, secondary antibodies (i) Alexa Fluor 488-conjugated goat anti-chicken IgG, (ii) Alexa Fluor 568-conjugated goat anti-mouse IgG2a, and (iii) Alexa Fluor 633-conjugated goat anti-rabbit (Molecular Probes) were diluted 1:500 in TBST and sections were incubated for 4h at room temperature. After washing 2×15 min with TBST and 1×15 min with TBS, sections were stained with DAPI, diluted 1:1000 in TBS. After several washes in TB, sections were mounted in Aqua-Poly-Mount (Polysciences).
For staining against oG, blocking solution was prepared with 3% bovine serum albumin/10% goat serum/TBST. Guinea pig anti-rabies glycoprotein antibody (kindly donated by A. Lüthi) was diluted 1:500 in blocking solution and combined with Alexa Fluor 633-conjugated goat anti-guinea pig IgG (Molecular Probes).

Hafner G., Witte M., Guy J., Subhashini N., Fenno L.E., Ramakrishnan C., Kim Y.S., Deisseroth K., Callaway E.M., Oberhuber M., Conzelmann K.K, & Staiger J.F. (2019). Mapping Brain-Wide Afferent Inputs of Parvalbumin-Expressing GABAergic Neurons in Barrel Cortex Reveals Local and Long-Range Circuit Motifs. Cell reports, 28(13), 3450-3461.e8.

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Immunostaining of Neural Stem Cells

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Wholemounts were incubated for 1 h in 0.5% triton-X in PBS (TPBS) then in 10% normal donkey serum (NDS; Jackson ImmunoResearch, 017000121) in TPBS for 1 h. Primary antibodies were incubated overnight at 4°C; for NSCs: chicken anti-GFP (1:100; Rockland, 600-901-215), rat anti-GFAP (1:500; Invitrogen, 13-0300) and for TAPs: rabbit anti-MASH1 (ASCL1) (1:100; Abcam, ab211327) in TPBS. After rinsing 3 times with TPBS, secondary antibodies (Jackson ImmunoResearch) were added at 1:300 in TPBS: donkey anti-chicken immunoglobulin G (IgG) 488 (703545155), donkey anti-rat IgG CY5 (712605153) (for NSCs), or donkey anti-rabbit (711605152) (for TAPs). Wholemounts were then washed 3 times with TPBS and incubated with DAPI (1:1,000; Invitrogen, D1306) for 10 min at room temperature (RT). After rinsing 3 times with TPBS, V-SVZs were mounted on glass slides with tissue spacers in Prolong Gold Antifade reagent with DAPI (Invitrogen, P366931).

Zhao X., Fisher E.S., Wang Y., Zuloaga K., Manley L, & Temple S. (2022). 4D imaging analysis of the aging mouse neural stem cell niche reveals a dramatic loss of progenitor cell dynamism regulated by the RHO-ROCK pathway. Stem Cell Reports, 17(2), 245-258.

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Fly Brain Dissection and Immunostaining

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Dissection of fly brains, fixation and staining of neuropil and neurons was performed as previously described 11 .
For primary antibodies, we used mouse anti-Brp (nc82, DSHB) at 1:10 and chicken anti-GFP (Rockland, 600-901-215) at 1:1000. For secondary antibodies, we used Alexa Fluor 488 goat anti-chicken (A11039, Invitrogen) at 1:800 and Alexa Fluor 633 goat anti-mouse (A21052, Invitrogen) at 1:400.

Lyu C., Abbott L.F, & Maimon G. (2022). Building an allocentric travelling direction signal via vector computation. Nature, 601(7891).

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Immunofluorescent Labeling of Cellular Proteins

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Cells were fixed for 15 min at room temperature in 4% (w/v) paraformaldehyde in PBS, washed 3 times in PBS, then permeabilized for 1 hr in Permeabilization buffer (PB: 0.3% Triton X-100, 0.3% BSA (Sigma), in PBS). Primary antibodies were incubated for 2 hr at room temperature in PB. Secondary antibodies were incubated for 1 hr at room temperature. Coverslips (BioCoat) were mounted on slides with Fluoromount G (EMS). Primary antibodies used for immunohistochemistry and immunocytochemistry in this study are chicken anti-GFP (Rockland) (1:2000) and anti-CRE (Millipore) (1:1000). All secondary antibodies were Alexa-conjugated (Invitrogen) and used at a 1:1000 dilution. Nuclear DNA was stained using Hoechst 33258 (1:10,000, Pierce).

Lanfranchi M., Meyer-Dilhet G., Reis R.D., Garcia A., Blondet C., Javin L., Amar A., & Courchet J. (2020). The AMPK-related kinase NUAK1 controls cortical axons branching though a local modulation of mitochondrial metabolic functions.

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Brain Tissue Immunostaining and Imaging

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At P21, mice were sacrificed by intracardiac perfusion of PFA (4% in PBS) followed by a 2 hours postfixation of the whole brain in a 4% PFA solution. 75µm thick sections were performed using a Leica VT1000S vibratome. Slices were permeabilized for 30 minutes in PB, then incubated overnight with primary antibodies (chicken anti-GFP, Rockland) diluted at 1:2000 in PB. The following day, we performed 3 washes in PBS 1X, then incubated slices in secondary antibody-containing PB (Goat antichicken antibody, Alexa 488, 1:2000, life technology). Nuclear DNA was stained using Hoechst 33258 (1:5000).

Lanfranchi M., Meyer-Dilhet G., Reis R.D., Garcia A., Blondet C., Javin L., Amar A., & Courchet J. (2020). The AMPK-related kinase NUAK1 controls cortical axons branching though a local modulation of mitochondrial metabolic functions.

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Immunohistochemical Analysis of mPFC

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Mice were anesthetized with 5% isoflurane, transcardially perfused with ice-cold PBS with 2g/L glucose followed by 4% PFA. Brains were dissected and post-fixed for 24hrs in 4% PFA at 4°C and then washed three times in PBS. Free-floating, 40-60 μm coronal mPFC sections were obtained using a Vibratome (Leica VT1200S, Leica Biosystems, Buffalo Grove, IL, USA) and stored in PBS until processing. Sections were washed with PBS, blocked with 5% normal donkey serum and 0.3% Triton X-100 in PBS for 2 hr at room temperature, and then incubated with primary antibody diluted in blocking buffer overnight at 4°C as follows: goat anti-RFP (Cat. No. 200-101-379, Rockland Immunochemicals, Inc., Limerick, PA) at 1:1000, chicken anti-GFP (Cat. No. ab13970, Abcam, Cambridge, MA, USA) at 1:2000. Slices were washed and then incubated with appropriate secondary antibodies for 2 hr at room temperature as follows at 1:500 dilutions: donkey anti-goat-Cy3 (Cat. No. 705-165-147) and donkey anti-chicken-Alexa488 (Cat. No. 703-545-155, Jackson ImmunoResearch Inc., West Grove, PA, USA). Slices were washed, incubated with DRAQ5 (Cat. No. 4084) for 5 min at room temperature, and then mounted and coverslipped onto Fisherbrand Superfrost Plus slides using Fluoromount Aqueous Mounting Medium. Sections were imaged as detailed for fluorescence in situ hybridization experiments.

Maksymetz J., Byun N.E., Luessen D.J., Li B., Barry R.L., Gore J.C., Niswender C.M., Lindsley C.W., Joffe M.E, & Conn P.J. (2021). mGlu1 potentiation enhances prelimbic somatostatin interneuron activity to rescue schizophrenia-like physiological and cognitive deficits. Cell reports, 37(5), 109950.

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9

Immunohistochemical Analysis of mPFC

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Mice were anesthetized with 5% isoflurane, transcardially perfused with ice-cold PBS with 2g/L glucose followed by 4% PFA. Brains were dissected and post-fixed for 24hrs in 4% PFA at 4°C and then washed three times in PBS. Free-floating, 40-60 μm coronal mPFC sections were obtained using a Vibratome (Leica VT1200S, Leica Biosystems, Buffalo Grove, IL, USA) and stored in PBS until processing. Sections were washed with PBS, blocked with 5% normal donkey serum and 0.3% Triton X-100 in PBS for 2 hr at room temperature, and then incubated with primary antibody diluted in blocking buffer overnight at 4°C as follows: goat anti-RFP (Cat. No. 200-101-379, Rockland Immunochemicals, Inc., Limerick, PA) at 1:1000, chicken anti-GFP (Cat. No. ab13970, Abcam, Cambridge, MA, USA) at 1:2000. Slices were washed and then incubated with appropriate secondary antibodies for 2 hr at room temperature as follows at 1:500 dilutions: donkey anti-goat-Cy3 (Cat. No. 705-165-147) and donkey anti-chicken-Alexa488 (Cat. No. 703-545-155, Jackson ImmunoResearch Inc., West Grove, PA, USA). Slices were washed, incubated with DRAQ5 (Cat. No. 4084) for 5 min at room temperature, and then mounted and coverslipped onto Fisherbrand Superfrost Plus slides using Fluoromount Aqueous Mounting Medium. Sections were imaged as detailed for fluorescence in situ hybridization experiments.

Maksymetz J., Byun N.E., Luessen D.J., Li B., Barry R.L., Gore J.C., Niswender C.M., Lindsley C.W., Joffe M.E, & Conn P.J. (2021). mGlu1 potentiation enhances prelimbic somatostatin interneuron activity to rescue schizophrenia-like physiological and cognitive deficits. Cell reports, 37(5), 109950.

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Chicken anti gfp

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9 protocols using chicken anti gfp

Unilateral Antennal Ablation and Fly Brain Dissection

Antibodies Used for Neuronal Immunostaining

Immunohistochemical Labeling of Barrel Cortex

Immunostaining of Neural Stem Cells

Fly Brain Dissection and Immunostaining

Immunofluorescent Labeling of Cellular Proteins

Brain Tissue Immunostaining and Imaging

Immunohistochemical Analysis of mPFC

Immunohistochemical Analysis of mPFC

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