Hsp60

Immunolocalization of HSP 90, 70, 60 and 27 was performed in all hemocyte cultures (controls, F24 and F48). The cells were fixed in 4% Paraformaldehyde (Sigma-Aldrich; PFA) in Phosphate-buffered saline (PBS) and permeabilized in 0.1% Triton X-100 (Sigma-Aldrich) in PBS. The cells were incubated overnight at 4°C with primary antibodies all from Enzo Life Sciences: HSP90α/β monoclonal antibody, HSP70 monoclonal antibody, HSP60 (insect) polyclonal antibody or HSP27 polyclonal antibody. Antibodies were diluted 1:40 in PBS with 1% bovine serum albumin (BSA, Sigma-Aldrich). The cells were then incubated in 4% BSA-PBS for two hours to prevent non-specific antibody binding. Following this, the hemocytes were incubated for a further two hours at room temperature with secondary antibody DyLight 488, Goat Anti-Mouse IgG (Abbkine) for HSP 90 and 70 or Dylight 594, Goat Anti-Rabbit IgG (Abbkine) for HSP 60 and 27. Concentrations of secondary antibodies were 2μg/ml. ActinGreen 488 ReadyProbes Reagent (Invitrogen) or ActinRed 555 ReadyProbes Reagent (Invitrogen) were used to label the actin fibers. The cell nuclei were stained with Hoechst (Enzo Life Sciences). Fluorescence signals were analyzed by fluorescent microscopy an Axio Vert.A1 fluorescence microscope (Zeiss) with Axio Cam ICc 5 (Zeiss).

Intrathecal injection into mice and analysis of the CSF were performed as described previously [7 (link),16 (link),19 (link)]. In detail, 40 μL of ligand solution (PCR studies after 12 h: 10 μg LPS, 10 μg Pam3CysSK4, 10 μg imiquimod, 10 μg CpG ODN; histologic studies after 72 h: 10 μg LPS, 40 μg Pam3CysSK4, 136 μg loxoribine, 10 μg CpG ODN, all obtained from Invivogen, or 10 μg HSP60 (Enzo Life Sciences) were injected intrathecally into 6- to 8-week-old male mice. Notably, the contamination with LPS of the HSP60 preparation as declared by the manufacturer was <50 EU/mg and ≤1.67 EU/mg, as determined by an independent laboratory specializing in endotoxin testing and published in previous work (Mikrobiologisches Labor, Münster, Germany; see also [18 (link)]). For real-time PCR analysis, brains were removed and cut along the sulcus medianus into two halves, which were separately snap-frozen in liquid nitrogen. For immunohistochemical studies, mice were perfused transcardially with isotonic-saline followed by 4% paraformaldehyde (PFA). Brains were removed, subjected to a row of 10%, 20%, and finally 30% sucrose in 0.1 M phosphate buffered saline (PBS) for cryoprotection, frozen in 2-methylbutan on dry ice, and stored at –80°C until sectioning.