Alexa fluor 488 or alexa fluor 594 conjugated anti mouse or anti rabbit secondary antibodies

Paraffin-embedded gastrocnemius and TA were cross-sectionally cut into 4-μm sections as above. After antigen retrieval, permeabilization, and goat serum blocking, primary antibody incubation was conducted overnight at 4 °C; primary antibodies included: mouse anti-MHC (1:1000), rabbit anti-TGF-β1 (1:100), rabbit anti-LC3B (1:200), rabbit anti-wheat germ agglutinin (WGA; 1:50; Abcam, USA) and rabbit anti-HMGB1 (1:400). Samples were then stained for 1 h with secondary Alexa Fluor 488- or Alexa Fluor 594-conjugated anti-mouse or anti-rabbit secondary antibodies (1:300, Invitrogen, USA), followed by a 5 min DAPI/PI (Sigma, USA) staining. Samples were then imaged using a fluorescence microscope. Similar procedures were carried out for C2C12 myotubes.

Western blot analysis using rabbit anti-SERA5, rabbit anti-AMA1 or the anti-MSP1 mAb X509 was performed as described previously (Jean et al., 2003 (link)). For IFA, thin blood films prepared from synchronous P. falciparum cultures enriched in mature schizonts were air dried, fixed with 4% (w/v) paraformaldehyde in PBS for 20 min and incubated in blocking buffer [10% (v/v) fetal calf serum, 3% (w/v) BSA, 0.5% (w/v) glycine in PBS] for 30 min at room temperature. Slides were probed with polyclonal rabbit anti-SERA5 (diluted 1:6000), anti-MSP1 mAb 89.1 ascites (diluted 1:1000) or purified anti-HA3 mAb 12CA5 (0.1 μg ml⁻¹), all diluted in blocking buffer containing 0.1% (w/v) saponin. The slides were washed in PBS 0.05% (v/v) Tween 20 (PBS-Tween) then bound primary antibodies detected using Alexa Fluor 488 or Alexa Fluor 594-conjugated anti-mouse or anti-rabbit secondary antibodies (Invitrogen) diluted 1:2000 in blocking buffer containing 0.1% (w/v) saponin. Samples were washed in PBS-Tween, stained with 4,6-diamidino-2-phenylindole (DAPI), mounted in Citifluor (Citifluor Ltd., Canterbury, UK), and images were captured using AxioVision 3.1 software on an Axioplan 2 Imaging system (Zeiss) using a Plan-APOCHROMAT 1006/1.4 oil immersion objective.