Axiocam hrm camera hal 100 lamp

As described before^{53 (link)}, adhered cardiomyocytes were fixed in 4% paraformaldehyde and washed with PBS-Tween 20 (PBS-T) twice and incubated with 0.1% sodium citrate and 0.05% triton-X100 for 10 min at RT. Non-specific sites were blocked using 3% normal horse serum (NHS) for 1 h and washed twice with PBS-T. Specific primary antibodies were used to detect LOX-1, troponin 1c and Cyt c with Alexa 488, 568 or 594 conjugated secondary antibodies (Invitrogen, USA). Expression and co-localization of these antibodies in cardiomyocytes were analyzed and quantified by microscopic analysis (Carl Zeiss., LSM 5 Pa, Germany) attached with Zeiss AxioCam HRM camera HAL 100 lamp, using AxioVision 4.8 software.

Tissue pieces from hearts after I/R with or without IL-10 were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue Sections (5–6 µm) were mounted on microscopic slides and de-paraffinized in xylene and hydrated through graded series of alcohol. Sections were washed with PBS twice and permeabilized in 0.05% triton-X100 for 15 min at RT. Tissue endogenous peroxidase was inhibited by antigen retrieval process using enzymatic retrieval buffer (10 mM Tris base, 1 mM EDTA, 10 mM sodium citrate and 0.05% Tween 20, pH 9.0). Fc receptor sites were blocked using 3% NHS for 1 h and washed twice with PBS-T. Specific primary antibody was used to detect TLR2 with Alexa 488 conjugated secondary antibody (Invitrogen, USA). Antibody to TGF-βRII was also used and stained with diaminobenzidine (DAB) followed by Hematoxylin/Eosin staining^{38 (link)}. Expression of these antibodies in cardiomyocytes was analyzed and quantified by microscopic analysis (Carl Zeiss., LSM 5 Pa, Germany) attached with Zeiss AxioCam HRM camera HAL 100 lamp, using AxioVision 4.8 software.

As described earlier [20 (link)], cardiomyocytes were allowed to adhere on chambered slides and fixed in 4% paraformaldehyde (PFA). Slides were washed with phosphate-buffered saline-Tween 20 (PBS-T) twice and permeabilized with 0.1% sodium citrate and 0.05% triton-X100 for 10 min at RT followed by 0.1% sodium borohydride treatment for 5 min at RT to quench autofluorescence. Slides were washed three times with PBS-T and incubated with 3% normal horse serum (NHS) for 1 h. Specific primary antibodies were used in co-localization of DDIT3 (Abcam, Cambridge, UK, cat #ab233121) together with iNOS, using Alexa 488, and 594 conjugated secondary antibodies (Invitrogen Life Technologies, Carlsbad, CA, USA). DAPI mounting media was used to locate nuclei. Slides were examined for co-expression of DDIT3/iNOS in Dox treated cardiomyocytes in the presence or absence of SNAP or L-NAME using the microscopic (Carl Zeiss., LSM 5 PASCAL, Jena, Germany) analysis setup attached with Zeiss AxioCam HRM camera HAL 100 lamp and quantified by using AxioVision 4.8 software.