Alexa fluor 555 goat anti mouse igm

Cells were fixed with 4% paraformaldehyde solution for 20 min at room temperature. After washing with PBS, the cells were permeabilized with PBS containing 0.1% Triton-X 100 and then blocked with blocking buffer (10% normal goat serum, and 0.1% Triton-X 100 in PBS) for 1 h at room temperature. The cells were then incubated with following primary antibodies, mouse anti-synaptophysin (1:200), mouse anti-PSD-95 (1:100), rabbit anti-MAP2 (1:100) for 4h at room temperature. After being washed with PBS, the cells were incubated with secondary antibodies for 2 h at room temperature. The secondary antibodies used were Alexa Fluor 488 goat anti-rabbit IgG (1:200, Invitrogen, CA, USA), Alexa Fluor 647 goat anti-mouse IgG (1:200, Invitrogen, CA, USA) and Alexa Fluor 555 goat anti-mouse IgM (1:200, Invitrogen, CA, USA). Then the cells were incubated in PBS containing 1 μM DAPI for 30 min at room temperature. The cells were then observed under a LSM 510 confocal microscope.

Serial sections were immunohistochemically stained for type I, IIa, IIx or IIb MHC using mouse monoclonal primary antibodies BA-D5 (supernatant; 1 µg ml⁻¹), SC-71 (supernatant; 1 µg ml⁻¹), 6H1 (supernatant; 10 µg ml⁻¹) and BF-F3 (supernatant; 5 µg ml⁻¹), respectively (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). One section was co-stained for type I, IIa and IIx MHC and a serial section for type IIb MHC.
Sections were fixed with ice-cold acetone for 15 min and then blocked for 45 min with 10% goat serum in phosphate-buffered saline (PBS) at room temperature. Following washing with PBS, the sections were incubated with the primary antibody for 90 min in a humid chamber. The sections were subsequently washed in PBS and incubated in the dark for 60 min with Alexa Fluor 350 goat anti-mouse IgG2_b (A-21140; 2 µg ml⁻¹, Invitrogen) and Alexa Fluor 488 goat anti-mouse IgG₁ (A-21121; 2 µg ml⁻¹, Invitrogen) for type I and IIa fibres, respectively, and Alexa Fluor 555 goat anti-mouse IgM (A-21426; 2 µg ml⁻¹, Invitrogen) for type IIx and IIb fibres. Sections were washed, dried and mounted using Prolong Diamond anti-fade mounting medium (Life Technologies). Sections without the primary antibodies served as negative controls. Images were taken with a Carl Zeiss Axio MRc Camera (Göttingen, Germany) on a Zeiss fluorescence microscope (10× objective).