Alexa fluor goat anti mouse igg1 specific 647

For all qualitative IF analysis of human brain tissue samples, freshly cut sections were prepared from late stage (Braak V–VI) AD and control patient tissues archived at the UNC Brain Bank. Paraffin embedded tissue blocks were sectioned at 7 µM thickness, adhered to slides, and deparaffinized in xylene. Slides were rinsed in ethanol and rehydrated (pure, 95%, 70%) in 1 X PBS. Sections were boiled for 10 min and incubated in hot Vectastain H-3300 citrate-based buffer for 30 min for antigen retrieval. Sections were then rinsed in 1X TBS, and permeabilized for 50 min in 2% TBS-Triton X-100 at RT. Blocking, primary, and secondary solutions matched the donor species of secondary antibodies used; sections were blocked in 1X TBS with 2% goat serum for 50 min. Sections were then incubated with primary antibody in 0.1% sodium azide solution for 48 h at RT, thoroughly rinsed in 1X TBS three times, and incubated in secondary antibody solution for 24 h at RT. DAPI staining was used to visualize nuclei. Primary antibodies and dilutions: Tau-1 (Millipore, MAB3420, 1:1000), GFAP 1:1000, (Dako, GA524). Secondary antibody dilutions: Alexa Fluor goat anti-mouse IgM μ-chain specific 488, 1:200 (Thermofisher, A-21042), Alexa Fluor goat anti-rabbit 568, 1:200 (Thermofisher, A-11011), Alexa Fluor goat anti-mouse IgG1 specific 647, 1:200 (Thermofisher, A-1240).

Mice were deeply anesthetized and transcardially perfused with 1X PBS and 15 mL 4% Paraformaldehyde. Brains were then post-fixed for 24–48 h in 4% PFA at 4 °C and cryoprotected in 30% sucrose solution before sectioning. Tissues were sectioned at 30–40 μM thickness and stored at −20 °C. Immunofluorescence staining was conducted using a free-floating technique, rinsing in 1X TBS between all major steps, gently agitated on a plate shaker. Sections were rinsed and permeabilized for 50 min in 2% TBS-Triton X-100 at RT. Blocking, primary, and secondary solutions matched the donor species of secondary antibodies used; sections were blocked in 1X TBS with 2% goat serum for 50 min each. Sections were then incubated with primary antibody in 0.1% sodium azide solution for 48 h at room temperature, thoroughly rinsed, and incubated in secondary antibody solution for 24 h at room temperature. DAPI staining was used to visualize nuclei. Primary antibodies and dilutions: Mouse IgM, kappa monoclonal MM-30, 1:1000 (Abcam, ab18401), AT8 1:500 (Thermofisher, MN1020), GFAP 1:1000, (Dako, GA524). Secondary antibodies and dilutions: Alexa Fluor goat anti-mouse IgM μ-chain specific 488, 1:200 (Thermofisher, A-21042), Alexa Fluor goat anti-rabbit 568 1:200 (Thermofisher, A-11011), Alexa Fluor goat anti-mouse IgG1 specific 647, 1:200 (Thermofisher, A-21240).