Mouse anti cd68

Fresh tissue and cultured cells were fixed in 4% paraformaldehyde, and immunostaining was performed using primary antibodies prepared in different species: rabbit anti-nestin (1:200, Chemicon International, Temecula, CA, USA), rabbit anti-Isl-1 (1:200, Chemicon International), mouse anti-Vimentin (1:500, Chemicon International), rabbit anti-PDX-1(1:500, Chemicon International), mouse anti-glucagon (1:1000, Sigma-Aldrich), guinea pig anti-insulin (1:100, Zymed Laboratories, South San Francisco, CA, USA), mouse anti-C-peptide (1:250, Chemicon International), rabbit anti-CD3 (ready to use, ZSGB-BIO, Beijing, China), mouse anti-CD4 (1:60, Dako, Glostrup, Denmark), mouse anti-CD8 (1:80, DAKO) and mouse anti-CD68 (1:80, DAKO). Binding of the primary antibody was visualized by either the immunofluorescence technique or an HRP enzymatic reaction using 3,3′-diaminobenzidine (DAB) as the chromogen. For Prussian blue staining, fixed cells and tissue sections were incubated in a 1:1 mixture of 4% potassium ferrocyanide and 4% HCl for 50 minutes. DAPI or nuclear fast red were used as nuclear counterstains. Representative images were captured using an Olympus BX53 Microscope or NIKON2000U microscope.

Tissue blocks containing infarcted human brain tissue were imbedded in paraffin and 2 μm thick, serial sections were cut on a microtome, deparaffinized, blocked in 1.5% H₂O₂ in Tris-buffered saline (TBS) and demasked in T-EG buffer (10 mM Tris + 0.5 mM EGTA, pH 9.0), whereafter they were loaded onto a Dako autostainer (Dako, Denmark)61 (link). Sections were stained using the following primary antibodies: rabbit anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:1,000, Wako), mouse anti-CD45 (1:200, Dako), mouse anti-CD68 (1:100, Dako), rabbit anti-GFAP (glial fibrillary acidic protein, 1:2,000, Dako), rabbit anti-TNF (1:100, Endogen)4 (link)23 (link)27 (link), rabbit anti-TNFR1 (1:50 (H-271), Santa Cruz), and rabbit anti-TNFR2 (1:50, Sigma-Aldrich). Secondary antibodies included EnVision + System horseradish peroxidase (HRP)-labelled Polymer (Dako) for CD45, CD68 and GFAP, Advance HRP (Dako) for TNF and TNFR1, and PowerVision + Poly-HRP IHC detection system (AH Diagnostics) for TNFR2. Omission of primary antibody and comparable concentrations of rabbit IgG were used to control for unspecific binding in the immunohistochemical protocols for TNF, TNFR1 and TNFR2 and these sections were devoid of staining. In addition, parallel sections were stained for hematoxylin and eosin (HE) using standard protocols at the Department of Pathology, Odense University Hospital.

The following antibodies were used at the indicated dilutions: mouse anti-CD68 (1:100, Daka, Agilent Technologies, Santa Clara, CA, USA, M0814), mouse anti-GFAP (1:800, Immunological Sciences, Roma, Italy, MAB12029) and rabbit anti-GRAMD1B (1:50, Sigma-Aldrich, HPA008557). The secondary antibodies used were anti-mouse Alexa 488 and anti-rabbit Alexa 546 (1:500, Thermo Fisher Scientific, A-11001 and A-11035, respectively).