Alexa488 conjugated goat anti mouse igg h l antibody

Biopsies of quadriceps muscles of 3 DMD patients and 2 normal controls were used (Table 1). Serial 7 μm cryosections were stained with H&E and an antibody against human CD133 (Milteni biotech, clone 293C3). For H&E staining, the sections were air dried before being stained with hematoxylin and eosin. The sections were dehydrated through 30%, 50%, 75%, 95% and 100% ethanol, followed by xylene for 10 min. The sections were mounted using DPX mounting solution. For immunostaining of CD133, muscle sections were air dried before being incubated in primary antibody (CD133, 1:100, diluted in PBS containing 10% normal goat serum and 0.03% Triton ×100) for 2 h at room temperature (RT). After rinsing with PBS 3 × 5 min, the sections were incubated with Alexa-488 conjugated goat anti-mouse IgG (H + L) antibody (1,1000, Thermo Fisher) for 1 h at RT. The sections were rinsed with PBS for 5 min and then mounted with DAKO mounting medium (containing 10 μg/ml DAPI).

Diluted mouse serum (1:100 in PBS) was incubated on HEp‐2 slides (Orgentech) for 30 min at 22°C, before the slides were washed twice for 5 min with PBS. For detection of mouse IgM or IgG, the slides were incubated for 30 min at 22°C with an Alexa488‐conjugated goat anti‐mouse IgM antibody or an Alexa488‐conjugated goat anti‐mouse IgG (H + L) antibody (both from Thermo Fisher Scientific) as a secondary antibody. Following two washing steps, DAPI‐containing mounting medium (Life Technologies) was added together with a cover slip. Images were acquired with a Zeiss Axio Imager 2 microscope and were analyzed with the Fiji software.