Chicken anti gfp

Adult male Drosophila brains were immuno-stained as described previously [55 (link)]. Briefly, brains were fixed in 4% paraformaldehyde for 20 min at RT, and blocked in 5% goat serum for 1 h at RT. Primary antibodies used were as follows: rabbit anti-DsRed (Clontech)– 1:2000; mouse anti-PDF (Developmental Studies Hybridoma Bank, DSHB)– 1:2000; mouse anti-Bruchpilot (nc82, DSHB)– 1:200; chicken anti-GFP (Invitrogen)– 1:1000 –rabbit anti-PER [58 ] 1:15000. Alexa-fluor secondary antibodies (goat anti-rabbit 555, goat anti-chicken 488, goat anti-mouse 488; Invitrogen) were used at 1:2000 except for labeling anti-BRP where goat anti-mouse 647 where a dilution of 1:500 was used. Confocal images were taken using an inverted Leica LSP8. PER signal intensity was quantified using an 40x oil-objective and imageJ. Pixel intensities were normalized to the background after background subtraction [(signal-background)/background] using Excel.

Immunohistochemistry follows protocols previously reported^{15 (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.

Mice were deeply anesthetized and perfused as previously described [11 (link)]. The brain was dissected, post-fixed in 4% PFA overnight, stored in PBS with 0.01% sodium azide, and sectioned at 50 μm using Leica vibratome (Leica VT1000S, Leica Inc., Nussloch, Germany). Floating sections were processed for immunocytochemistry [10 (link)]. All antibodies were obtained from Abcam (Cambridge, MA, USA) unless otherwise stated. In this study, chicken anti-GFP (1:500), rat anti-GFAP (1:1000; Invitrogen), rabbit anti-Iba1 (1:500; Wako), TMEM 119 (1:500), mouse anti-CCR2 (1:500), and rabbit anti-CD31 (1:500) were used.
For postmortem human samples, slides were baked for 60 min at 60 °C, deparaffinized with xylene for 5 min, and rehydrated in ethanol (100, 95, 70, and 50%). Antigen retrieval was performed as previously reported [17 (link)]. In this study, chicken anti-Map 2 (1:200, Abcam, Cambridge, MA, USA), rat anti-GFAP (1:1000; Invitrogen, Thermo Fisher Scientific, Rockford, IL, USA), rabbit anti-Iba1 (1:500; Wako), TMEM 119 (1:1000), mouse anti-CCR2 (1:500), and rabbit anti-CD31 (1:500) were used.

For western blotting, the brain and ventral nerve chord of larval fillets were removed and subjected to different stimulation conditions. Afterwards, the fillets were crushed in 1xSDS sample buffer and boiled for 5 min. Dilutions for primary antibodies were as follows: mouse anti-α-actin, 1:20000 (Sigma); chicken anti-GFP, 1:5000 (Invitrogen).

VGF was overexpressed in hippocampal cell cultures using adeno-associated virus (AAV) vectors tagged with GFP (Vector BioLabs, Eagleville, PA, USA). Hippocampal neuronal cultures were treated with VGF-GFP or control GFP virus at 14 DIV. Each culture dish was treated with 20 µL of either virus (VGF-GFP or GFP) for 6 days at final concentrations of 2.45 × 10⁹ GC/mL for the VGF-GFP virus and 9.80 × 10⁸ for the GFP control virus in Neurobasal medium. Dishes were then fixed in 4% PFA at 20 DIV. Immunostaining was performed in order to enhance the GFP fluorescence. Cells were incubated in chicken anti-GFP (1:500, Invitrogen) overnight at 4 °C. Secondary antibody, rabbit anti-chicken FITC (1:500, Invitrogen) was applied for 1 h at room temperature. The dishes were slipped with Fluoromount-G. Spine morphology observation was performed with a confocal microscope with the Fluoview program. Dendrites were imaged with a 60× lens and 2× digital zoom. Spines were measured in ImageJ software for head width and spine length. Head width was measured as the distance from the rightmost side of the largest part of the spine to the leftmost side. Spine length was measured as the distance from the top of the spine to its base. For dendritic spine density, spines were counted per 20 µM dendritic segment in Axiovision software (Version 4.8.2, Zeiss, Jena, Germany).