Ab7207

After dewaxing in xylene and rehydration in graded alcohols, the tissue sections were boiled in citrate buffer, pre-incubated with H₂O₂, and then blocked with rabbit or goat serum (DAKO, Denmark). Subsequently, the sections were incubated with a primary antibody and then with an HRP-conjugated secondary antibody. The proteins of interest were visualized by using diaminobenzidine before counterstaining with hematoxylin. The primary antibodies used were as following: MIF (ab7207, Abcam, USA), CD68 (ab283654, Abcam, USA) and HIF-α (ab51608, Abcam, USA).

The cells were fixed in 4% PFA for 10 min and washed three times in washing buffer. After washing three times in washing buffer (PBS + 0.25% Triton X-100 + 025% BSA), the slides containing astrocytes were blocked in blocking solution (PBS + 0.25% Triton X-100) for 30 min at room temperature. Incubation with the corresponding antibodies diluted in blocking solution was done overnight at 4 °C. The following primary antibodies were used: primary polyclonal rabbit anti-MIF antibody (1:200; Abcam ab7207), primary polyclonal rabbit HTRA1 (1:200, A kind gift from Michael Ehrmann at Universität Duisburg-Essen, Essen, Germany), primary polyclonal chicken anti-GFAP (1:1.000; Abcam, ab4674), polyclonal rabbit anti-FGF8 (1:200, Abcam, ab81384) and polyclonal mouse anti-FGF18 (1:200, Abcam, ab169615). Incubation with the secondary antibodies was performed in the dark at room temperature for 1 h. The secondary antibodies were as following: Alexa Fluor-488 goat anti-rabbit IgG (1:400; Invitrogen, 828814), Rhodamine Red-X goat anti-chicken (1:400; Jackson Immuno Research, 93951) and Rhodamine Red-X goat anti-mouse (1:400; Jackson Immuno Research, 94085). The slides were washed in PBS and stained with DAPI for 20 min before mounting. For all staining procedures, negative control antibodies were performed.

For immunofluorescence experiments [23 (link),26 (link)], cells were processed as previously described, with slight modifications [23 (link),26 (link)]. Briefly, THP-1 macrophages were incubated for 1 h and immediately fixed with pre-warmed 3.7% formaldehyde (15 min, room temperature). After permeabilization (0.2% Triton-X-100, 10 min), unspecific binding sites were blocked (2% Donkey Serum, 0.5% Bovine Serum Albumine, 1 h). Afterwards, cells were incubated for 2 h with anti-Caveolin-1 antibody (Abcam, ab192452, 1:500), anti-TLR4 antibody [76B357.1] (Abcam, ab22048, 1:200) and anti MIF antibody (Abcam, ab7207, 1:500). After 5 washing steps, species-specific fluorescence-labeled antibodies were used for localization (Donkey Anti-Goat IgG (H+L), Alexa Fluor^® 647 (705-605-003) and Donkey Anti-Mouse IgG (H+L) Alexa Fluor^® 488 (715-545-150) from Jackson ImmunoResearch Laboratories, Inc., Pennsylvania, US and Donkey anti-Rabbit IgG (H+L) polyclonal secondary antibody, Alexa Fluor^® 568 (A10042) from Thermo Fisher Scientific, Waltham, US).