Donkey anti mouse igg

Tissue sections were immunostained as previously described [28 (link)]. Primary antibodies included rabbit anti-Ki67 (Thermo Fisher Scientific, Inc., Fremont, CA; Cat. No. RM-9106-S1) (1:200), mouse anti-BrdU (Thermo; Cat. No. MS-1058-PO) (1:250), goat anti-cryptdin related sequence 4C (CRS4C) (gift from Dr. A. J. Ouellette, University of Southern California, Los Angeles, CA) [32 (link)] (1:2000), and rabbit anti-MUC2 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA; Cat. No. sc15334) (1:100). Secondary antibodies included biotinylated donkey anti-rabbit IgG, donkey anti-goat IgG, and donkey anti-mouse IgG (all from Vector Labs, Burlingame, CA). Biotinylated antibodies were linked to avidin-horseradish peroxidase conjugates (Vector Labs), visualized using 3,3′-diamino benzidine (Sigma) for 2 to 5 min, and lightly counterstained with hematoxylin.

To evaluate the permeability of the blood–brain barrier (BBB), sections were incubated in 3% H₂O₂, blocked with 10% bovine serum albumin (Sigma‐Aldrich) in PBS, and incubated overnight at 4°C with donkey anti‐mouse IgG (1:300; Vector Laboratories). Immunoreactivity was visualized using the avidin‐biotin complex method (Vectastain ABC kit, Vector Laboratories) and quantified using ImageJ.

Twenty-four hours after reperfusion, mice were anesthetized with pentobarbital (50 mg/kg, i.p.) and decapitated. The brains were carefully removed and fixed in 4% paraformaldehyde for at least 2 days at 4°C and then overnight in 30% sucrose. To evaluate the permeability of the BBB to endogenous IgG proteins, we assessed coronal cryostat brain slices (20 μm) that were stained with IgG antibodies (donkey anti-mouse IgG, 1:300; Vector Laboratories). Immunoreactivity was visualized using the avidin–biotin complex method (Vectastain ABC kit, dilution 1:400; Vector Laboratories) [34 (link)], scanned with AxioVision software (Carl Zeiss MicroImaging GmbH, Göttingen, Germany), and measured using the ImageJ program (NIH, http://rsb.info.nih.gov/nih-image/). We measured the total luminances of the ipsilateral and contralateral hemispheres from each slice, and then computed an average luminance for each mouse. The number of mice in each treatment group are indicated for each result. IgG staining was then calculated as follows: (average of total ipsilateral hemispheric luminance − average of blank luminance) / (average of total contralateral hemispheric luminance − average of blank luminance).