Skip to main content
. Author manuscript; available in PMC: 2013 May 8.
Published in final edited form as: Exp Parasitol. 2012 Oct 22;133(1):28–36. doi: 10.1016/j.exppara.2012.10.002

Figure 2. Immunocytochemical (ICC) and Western (−LTP)/far-Western (+LTP) blot analyses of hemocyte-associated glycotopes recognized by mABs binding to (A) trimannosyl core N-glycan [TriMan; Man(α1–6)[Man α1–3)]Man(β1–4)GlcNAc(β1–4)GlcNAc], and the fucosylated lacdiNAc variants (B) F-LDN-F; Fuc(α1–3)GalNAc(β1–4)[Fuc(α1–3)]GlcNAc and (C) F-LDN; Fuc[(α1–3)]GalNAc(β1–4)GlcNAc.

Figure 2

The ICC images of hemocytes (right panel) showing the heterogeneity of staining intensities and cell-types exhibiting specific glycotope reactivities within a single hemocyte population of both B. glabrata snail strains. TriMan-associated glycans are distributed mainly on granulocyte-type cells and substrate-adherent plasma proteins, although small spread cells (Fig. 2A; arrows) and distinctive rounded hemocytes (type “B” hyalinocyte [Schoenberg and Cheng, 1980; Yoshino and Granath, 1994]; Fig. 2A, arrowheads) completely lack TriMan glycotope expression. In contrast, granulocytes staining with F-LDN and F-LDN-F mABs were highly variable, ranging in reactivities from intensely fluorescent to negative (Figs. 2B, 2C; arrows). Moreover, the TriMan-negative type “B” hyalinocytes exhibited strong F-LDN-F mAB staining (Fig. 2B; arrowheads). Hemocyte treatment with LTPs had no effect on the prevalence or cellular distribution of glycotopes in either snail strain. Western blot analyses of shared glycotopes associated with hemocyte proteins (–LTP; left blot panels) showed a similar banding profile for TriMan glycotopes between NMRI (N) and BS-90 (B) snail strains, with the exception of bands between 15–20 kDa (Fig. 2A; arrows). A greater intensity and distribution of F-LDN-F (Fig. B) and F-LDN (Fig. C), however, was noted for N B. glabrata hemocyte proteins compared to the B snail strain. Using a far-Western blot approach, additional F-LDN-F and F-LDN-reactive bands (15–37 kDa range) were seen in blots exposed to LTP (“+LTP”, right blot panels) prior to specific mAB exposure. Arrows in Figs. 2B and 2C (“+LTP” panels) indicate appearance of reactive bands unique to the B snail strain.