Alexa fluor 488 conjugated anti mouse igg a11029

Thin sections of paraffin-embedded tissues (brain and pancreas) were de-paraffinized, blocked in blocking solution (10% goat serum+5% BSA+0.5% triton X-100) and then incubated with primary antibodies at 4°C overnight. In the immunofluorescence measurements, a combination of anti-amylin (SC-377530; Santa Cruz biotech; TX, raised in mouse) and anti-4-HNE (ab46545; Abcam; MA, raised in rabbit), anti-MDA (ab94671; Abcam; MA, raised in rabbit), or anti-IL-1β (ab9722; Abcam; MA, raised in rabbit) primary antibodies were used. After washing, sections were incubated with Alexa Fluor 488 conjugated anti-mouse IgG (A11029; Invitrogen; NY) and Texas red conjugated anti-rabbit IgG (SC-2780; Santa Cruz biotech; TX) secondary antibodies. The sections were then stained with DAPI (ab 104139, Abcam; MA) and imaged with a laser-scanning confocal microscope (Live5; Zeiss; Germany). Immunofluorescence measurements were also done on fixed neurons incubated with anti-IL-1β primary and Alexa Fluor 488 conjugated secondary antibodies. Immunofluorescence staining for amylin was verified with a second anti-amylin antibody (T-4157; Bachem-Peninsula Laboratories; San Carlos; CA, raised in rabbit; Supplementary Figure 1). Tissue autofluorescence and elastin autofluorescence were blocked with 1% Sudan black (Supplementary Figure 2).

We used brain tissues from humans, HIP, WT and AKO rats, and isolated brain capillaries from HIP, WT and AKO rats. Brain tissue was processed as previously^{29 (link)} described. Brain capillaries were isolated (as described above) and then plated on laminin (23017-015, Gibco, MA) coated chambered coverglass for 1-hour, to allow time to attach.
Antibodies against human amylin (1:200, SC-377530, Santa Cruz, TX), collagen IV (1:500, SAB4200500, Sigma, MO), caveolin1 (1:1,000, sc-894, Santa Cruz, TX) and APOE (1:200, ab183597, Abcam, MA) were the primary antibodies. Alexa Fluor 488 conjugated anti-mouse IgG (A11029, Invitrogen, MA) and Alexa Fluor 568 conjugated anti-rabbit IgG (A11036, Invitrogen, MA) were the secondary antibodies.