9000 s microtome

Methods for tissue fixation, preparation of free-floating sections using a Leica 9000 s microtome (Leica, Wetzlar, Germany) and subsequent immunofluorescence staining were performed as described previously (Kowalewski et al., 2015 (link)). Primary antibodies used for immunofluorescence were against GFAP (1:500; clone G-A-5, G3893, Sigma); NeuN (1:2000; clone A60, MAB377, Millipore, Merck, Darmstadt, Germany), Olig2 (1:1000; AB9610, Millipore), Iba1 (1:500; GTX100042, GeneTex, Irvine, CA, USA), CD68 (1:500; clone FA-11 (MCA1957, AbD Serotec), calbindin (1:500; D-28K clone CB-955, C9848, Sigma), myelin basic protein (1:1000; MAB386, Millipore), Lamp1 (1:500; clone 1D4B, see above). The monoclonal antibody against GM2 gangliosides (IgM from mouse) was a kind gift from Prof. Kostantin Dobrenis (Albert Einstein College of Medicine of Yeshiva University, NY). Secondary antibodies were Alexa-Fluor conjugates (1:2000) and purchased from Invitrogen. Nuclei were stained with DAPI (Sigma). Confocal microscopy was performed with an LSM 700 (Zeiss, Oberkochen, Germany) or Olympus FV1000 microscope (Tokyo, Japan).

Methods for tissue fixation, preparing of free-floating sections using a Leica 9000s microtome (Leica, Wetzlar, Germany) and subsequent immunofluorescence staining were performed as described previously (Kowalewski et al., 2015 (link)). Primary antibodies used by immunofluorescence: glial fibrillary acidic protein-GFAP (1:500; clone G-A-5, G3893, Sigma); NeuN (1:1000; clone A60, MAB377, Millipore, Merck, Darmstadt, Germany), CD68 (1:500; clone FA-11 (MCA1957, AbD Serotec), myelin basic protein (1:500; MAB386, Millipore), Lamp1 (1:500; clone 1D4B, Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA)). Monoclonal antibody against GM2 gangliosides (IgM from mouse) was a kind gift from Prof. Kostantin Dobrenis. Secondary antibodies: AlexaFluor-coupled antibodies (1:2000) were purchased from Invitrogen. Nuclei were stained with DAPI (Sigma). Confocal microscopy was carried out with a LSM 700 (Zeiss, Oberkochen, Germany) or Olympus FV1000 microscope (Tokyo, Japan).