Anti gfap cy3

To visualize tissue properties, the sections were stained with hematoxylin and eosin (H&E).
Sections were washed in a. dest and incubated in Meyer's hematoxylin/hemalum (Sigma Aldrich, Steinheim, Germany) for 3 min. After washing in a. dest the tissue was briefly destained in HCl-ethanol. Washing using tap water for 5 min was followed by 3 min staining in eosin (1% eosin G in 80% ethanol). The sections were dehydrated with rising ethanol concentrations, cleared in xylene and coverslipped using DePex. For immunohistochemistry, sections were fixed in Methanol-Acetone at −20°C for 10 min followed by heat antigen retrieval in citrate buffer. After blocking in 5% bovine serum albumine in 0.3% TritonX for 1 h the tissue was probed with the primary antibody for 1 h (Anti-GFAP-Cy3, 1∶800, Sigma Aldrich). After washing with PBS, the sections were mounted with Vectashield containing DAPI (Vector Laboratories Inc., Burlingame, CA, USA).

Heat antigen retrieval in citrate buffer was performed followed by 0.3% TritonX for 15 min and blocking in 5% bovine serum albumin in PBS for 1 h. For GFAP immunohistochemistry, the tissue was probed with the antibody (Anti-GFAP-Cy3, 1:200, Sigma Aldrich) overnight at 4 °C. Rabbit anti- β-III-tubulin (1:500, Covance Inc., Princeton, NJ, USA), rabbit anti-Iba1 (1:200, Wako Chemicals GmbH, Neuss, Germany) and rabbit anti-GAP43 antibody (1:250, abcam, Cambridge, UK) were incubated overnight at 4 °C. After washing with PBS, sections were probed with fluorescent secondary antibodies (donkey anti-rabbit Alexa Fluor 594 (1:500, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. Stained fluorescent structures were visualized using a microscope Axio Examiner Z.1 equipped with camera AxioCam and metal halide lamp HXP 120c (Carl Zeiss AG, Jena, Germany), or by two-photon fluorescence using multiphoton microscopy.

The antibody catalogue numbers and the concentrations used for immunoblotting (IB), immunocytochemistry (ICC) and flow cytometry (FC) are provided in parentheses. Goat polyclonal anti-Hsp27/Hsp25 (sc-1049; IB 1:1000, ICC 1:100, FC 1:100) was from Santa Cruz Biotechnology. Rabbit polyclonal anti-GFAP (ab7260; IB: 1:10,000, ICC: 1:1000) and β-actin (ab8226; IB: 1:40,000) was from Abcam. Mouse monoclonal anti-α-tubulin (A11126; IB: 1:1000) was from Thermo Fisher Scientific. Rabbit polyclonal anti-Iba1 (019-19741, IB 1:5000, ICC 1:500) was from Wako Laboratory Chemicals and rat polyclonal anti-CD11b-APC/Cy7 (101226, FC 1:1000) was from Biolegend. Mouse monoclonal anti-GFAP-Cy3 (C9205, ICC 1:500, FC 1:500) was from Sigma-Aldrich. Donkey anti-rabbit IgG-AlexaFluor488 (A21206, ICC 1:1000), anti-goat IgG-AlexaFluor488 (A11055, ICC 1:1000, FC 1:1000) secondary antibodies were from Thermo Fisher Scientific. Rabbit anti-mouse IgG-HRP (P0260, IB 1:1000), anti-goat IgG-HRP (P0160, IB 1:2000), and pig anti-rabbit-HRP (P0217, IB 1:2000) secondary antibodies were from Dako-Agilent Technologies.