Alexa fluor 488 donkey anti chicken

To evaluate the presence of neuroinflammation after peripheral LPS treatment, prefrontal coronal sections were tagged for glial fibrillary acidic protein (GFAP) or ionized calcium binding adaptor molecule (Iba-1) costained with CD68 as astrocyte and microglial markers, respectively. Free-floating sections were rinsed of cryoprotectant solution, incubated in antigen unmasking solution (Vector, Burlingame, CA, USA) at 95°C for 7 min, and rinsed 3 × 5 min in 1× PBS. Sections were blocked in 10% donkey serum, 0.3% Triton, and PBS for 2 h and incubated overnight in primary antibody solution, including 1% bovine serum albumin (BSA), 0.3% triton, and PBS (chicken anti-GFAP: 1:500, ABCam, Cambridge, MA, USA; goat anti-Iba-1: 1:500, ABCam; mouse anti-rat CD68: 1:200, BioRad, Hercules, CA, USA). The sections were rinsed and incubated for 2 h in secondary antibody solution of 1% BSA in PBS (Alexa Fluor 488 donkey anti-chicken, 1:500, Jackson, Bar Harbor, ME, USA; Alexa Fluor 488 donkey anti-goat, 1:500, Life Technologies, Waltham, MA, USA; Alexa Fluor 594 donkey anti-mouse, 1:800, Life Technologies, Waltham, MA, USA). After a final rinse, slices were mounted onto gelatin-coated slides and coverslipped with mounting medium containing DAPI (Vector).

Mice were anesthetized with phenytoin/pentobarbital and perfused with phosphate-buffered saline (PBS; pH 7.4), followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH = 7.4). After an overnight post-fix in 4% PFA, brains were cryoprotected overnight in 30% sucrose before being embedded in OCT and stored at −80 °C. Free-floating sections (30 µm) were prepared with a cryostat. They underwent three washes in PBST (PBS + 0.3% Triton X-100) and were blocked in 3% normal donkey serum in PBST (1–2 h). Sections were then incubated overnight at 4 °C in primary antibodies, including rabbit anti-Satb2 1:2500 (ab34735, Abcam), mouse anti-Satb2 1:1500 (ab34735, Abcam), rabbit anti-Fos 1:2000 (#22505, Cell Signaling; ab190289, Abcam), goat anti-Fos 1:300 (sc-52-G, Santa Cruz), chicken anti-GFP 1:10,000 (ab13970, Abcam), goat anti-CGRP 1:1000 (ab36001, Abcam), and/or rabbit anti-dsRed 1:1000 (632475, Takara). The next day, the tissue was washed three times in PBS and incubated 1–2 h in appropriate secondary antibodies including Alexa Fluor 488/594 donkey anti-sheep, Alexa Fluor 488/594 donkey anti-goat, Alexa Fluor 488/594 donkey anti-rabbit, or Alexa Fluor 488 donkey anti-chicken (1:500; Jackson Immunoresearch Laboratories). Tissue was washed three times in PBS, mounted onto glass slides, and coverslipped with Fluoromount-G (Southern Biotech).

Immunohistochemistry, imaging, and quantification were performed as previously described [40 (link)]. Primary antibodies included rabbit anti-CSF1 (ab233387; Abcam), rabbit anti-Cre (69050; Novagen), or chicken anti-eGFP (ab13970; Abcam). Secondary antibodies included Alexa Fluor 488 donkey anti-rabbit (Jackson ImmunoResearch, 711-545-152), Alexa Fluor 555 donkey anti-rabbit (A32794, Invitrogen), or Alexa Fluor 488 donkey anti-chicken (703-545-155, Jackson ImmunoResearch).

Mice were anesthetized with Beuthansia (0.2 ml, i.p.; Merck) and perfused transcardially with PBS followed by 4% PFA in PBS. Brains were post-fixed overnight in 4% PFA at 4 °C, cryoprotected in 30% sucrose, frozen in OCT compound (ThermoFisher), and stored at −80 °C. Coronal sections (30 μm) were cut on a cryostat (Leica Microsystems) and collected in cold PBS. For immunohistochemistry experiments, sections were washed three times in PBS with 0.2% Triton X-100 (PBST) for 5 min and incubated in blocking solution (3% normal donkey serum in PBST) for 1 h at room temperature. Sections were incubated overnight at 4 °C in blocking solution with primary antibodies including chicken-anti-GFP (1:10000, Abcam, ab13970) and rabbit-anti-dsRed (1:2000, Takara Bio, 632496). After 3 washes in PBS, sections were incubated for 1 h in PBS with secondary antibodies: Alexa Fluor 488 donkey anti-chicken, Cy5 donkey anti-chicken, Alexa Fluor 594 donkey anti-rabbit, and/or Cy5 donkey anti-rabbit (1:500, Jackson ImmunoResearch). The tissue was washed 3 times in PBS, mounted onto glass slides, and coverslipped with Fluoromount-G (Southern Biotech). Fluorescent images were acquired using a confocal microscope. All digital images were processed in the same way between experimental conditions to avoid artificial manipulation between different datasets.

Kidneys were fixed in 4% PFA overnight at 4 °C, washed with PBS, embedded in paraffin blocks, 5 µm sections were generated and blocked in 10% normal donkey serum in PBS containing 0.3% Triton-X (blocking buffer) at room temperature for 1 h. Primary antibodies used included chick anti-GFP (1:250; Aves Labs), and rabbit anti-phospho-S6 ribosomal protein Ser240/244 (1:500; Cell Signaling Technology, Danvers, MA, USA, Catalog #5364). Slides were incubated with primary antibody in blocking buffer overnight at 4 °C, and washed 3 times in PBS with 0.1% Tween-20 for 20 min at room temperature. After washing, slides were incubated with secondary antibody (Alexa Fluor 488 donkey anti-chicken; 1:250; Jackson ImmunoResearch, Catalog #703-545-155 and Cy3 donkey anti-rabbit IgG; 1:250; Jackson ImmunoResearch) at room temperature for 1 h followed by washing and DAPI staining at 4 °C. Slides were mounted in Prolong Gold Antifade Reagent (Cell Signaling Technology) and imaged with a confocal Nikon A1R inverted microscope with a 20X objective lens using a pinhole of 1.2 μm on all channels at a resolution of 1024 × 1024.

TH-GCaMP5 transgenic mice (n = 3) were deeply anesthetized via inhalation of isoflurane and transcardially perfused with ice-cold saline followed by 10% formalin. Brains were harvested, postfixed in 10% formalin for 24 h, and then transferred into 30% sucrose for cryopreservation until sectioning (30 µm per section) throughout the brain using a sliding microtome. Brain sections were processed using free-floating immunohistochemistry to analyze the coexpression of TH in neurons expressing the calcium indicator GCaMP. Sections were blocked in 3% normal donkey serum, followed by incubation in primary antibodies, rabbit anti-tyrosine hydroxylase (1:1,000, AB-152; Millipore) and chicken anti-GFP (1:1,000, ab-13970; Abcam; GFP is a component of GCaMP5), and detected with fluorescent-labeled secondary antibodies Alexa Fluor 594 donkey anti-rabbit (1:200, A-21207; Thermo Fisher Scientific) and Alexa Fluor 488 donkey anti-chicken (1:200, 703-546-155; Jackson ImmunoResearch Laboratories), respectively. Histological sections from each mouse at the level of the area postrema were examined microscopically. Cells within the NST demonstrating antibody label for TH, GFP, or both labels were counted and photographed.