Cy3 donkey anti mouse igg

For the visualization of replicating DNA and epigenetic marks of heterochromatic blocks, female Microtus cabrerae cells were transiently transfected with CFP-PCNA and were pulse labeled for 20 min with 10 µM 5′-ethynyl-2′-deoxyuridine (EdU) (Invitrogen, Carlsbad, USA, Cat #: C10337) followed by a 1-h chase prior fixation with 4% paraformaldehyde. EdU was detected with the ClickIT system (Invitrogen) and AlexaFluor 488 followed by an immunostaining with rabbit anti-H3K27me3 (1/200, Upstate, Lake Placid, USA, Cat #: 07-449) and with mouse anti-H3K9me3 (1/100, Active Motif, Carlsbad, USA, Cat #: 39285). The following secondary antibodies were used: donkey anti-mouse IgG-Cy3 (1/200, The Jackson Laboratory, Bar Harbour, USA, Cat #: 715-166-151) and donkey anti-rabbit IgG-Cy5 (1/200, The Jackson Laboratory, Bar Harbor, USA, Cat #: 711-175-152). DNA was counterstained with 1 µg/ml DAPI for 10 min at RT, and cells were mounted afterward in Vectashield antifade (Vector Laboratories) (Fig. 2).

After a 4 hour Chandler loop rotation, blood was drawn out of the loops and the loops were flushed with 30 ml of PBS to remove excessive cells which were not firmly adhering to the grafts surfaces. BNC grafts were cut and embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, NL) and stored at -80°C. After freezing, cryosectioning was performed using a Leica CM 1950 cryostat (Leica Biosystems, Nussloch, GER). Here, activated thrombocytes were detected using rabbit anti-human CD62P (Biorbyt #orb416329) as primary antibody and respective donkey anti-rabbit Alexa Fluro 488 (Jackson ImmunoResearch #711-547-003) as secondary antibody. Likewise, erythrocytes were detected using mouse anti-human CD235a (Biorbyt #orb248903) as primary antibody and respective donkey anti-mouse IgG Cy3 (Jackson ImmunoResearch #715-167-003) as secondary antibody. Upon acetone fixation, the graft slices were blocked with 3% standard donkey serum and then incubated with the primary antibodies at the dilution of 1:50 in 3% donkey serum overnight. After washing, the fluorescence dye conjugated secondary antibody at the dilution of 1:500 in 3% donkey serum was incubated on the slice in the dark for one hour. The slices were then counterstained with DAPI (Sigma D9542) to stain the nuclei before mounting and microscopy (EVOS Auto 2, ThermoFisher Scientific, Massachusetts, USA).