Immpress anti mouse igg

To obtain autoradiographic information at the cellular resolution level, frozen cryostat sections, adjacent to those used for phosphor screen autoradiography, were coated with a liquid photographic emulsion following our previously published protocol [5 (link), 9 (link), 22 (link), 34 (link)]. Immunohistochemistry was then performed on the nuclear emulsion-dipped sections. First the sections were washed for 5 min with PBS, then incubated with 2.5% normal horse blocking serum for 20 min, followed by the appropriate primary antibody - anti-tau PHF-1 (1:100, mouse, kind gift of Dr. Peter Davies), anti-Aβ (1:500, mouse, clone 6F/3D, Dako), anti α-synuclein (1:100, mouse, Zymed) or anti-phospho TDP-43 (pS409/410) (1:3000, mouse, Cosmo Bio CO) - for 40 min at 37 °C, washed with PBS twice for 2 min, and then incubated with the secondary antibody (ImmPRESS™ anti-mouse IgG (Vector Laboratories product MP-2400, Burlingame, CA) or ImmPRESS™ anti-rabbit Ig (Vector Laboratories product MP-7401, Burlingame, CA)) for 40 min at 37 °C. The sections were washed again with PBS twice for 2 min, and developed with DAB solution (Vector Laboratories product SK-4100). H&E was used for counterstaining. Photomicrographs were obtained on an upright Olympus BX51 (Olympus, Denmark) microscope using visible light.

Four microm thick sections, which had been stored in 4% formaldehyde, were deparaffinized and rehydrated. The sections kept in citrate buffer, were heated in microwave for 15 min. The sections were then incubated with Formic acid 80% for 10 minutes, prior to blocking in a Peroxidase Block, 3% H2O2 for 10 minutes.
Following brief washes with Blocking buffer (5% goat serum, 5% BSA) for 30 minutes, the sections were incubated with Primary Antibody 6E10 (1:1000), a mouse monoclonal antibody, APP (Biolegend, MA, USA), and incubated over night at -4°C. The next day the slices were incubated with ImmPRESS (anti-mouse IgG, Vector Laboratories, CA, USA) for 30 minutes. For visualization DAB plus Peroxidase Substrate (Vector Laboratories, CA, USA) Kit were used.

After counting the number of blood vessels, CAMs were harvested, fixed, and embedded in paraffin for histological examination. As described previously [24 (link)], deparaffinized sections were subjected to hematoxylin-eosin (HE) staining and Masson’s trichrome staining.
Alternatively, the sections were antigen-retrieved and blocked with 2.5 % normal horse serum (Vector Labs, Burlingame, CA) and subsequently probed with a mouse monoclonal anti-α-smooth muscle actin (α-SMA) antibody (1:100) (Abcam, Cambridge, MA, USA) overnight at 4 °C, followed by incubation in the ImmPRESS® anti-mouse IgG (Vector). Immunoreactive proteins were visualized by a DAB substrate solution (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD).
The number of α-SMA⁺ vessel-like structure (shown by arrows) inside the CAM was determined using image analysis software (WinROOF, Mitani Corp., Fukui, Japan). In brief, after the contrast of the images was enhanced manually, the number of vessels was counted. The area of the CAM in the cross-sections was also evaluated. Then, the number of vessels was normalized by utilizing the ratio of the area to the number of vessels per unit square (mm²).