Lsm 5 pascal model confocal microscope

Liver morphology was evaluated through formalin-fixed paraffin-embedded tissue staining. Paraffin kidney sections (5 μm thick) were routinely stained for microscopic evaluation with H&E (Merck) or Sirius Red for collagen detection.
Liver fibrosis was quantified by measuring collagenous fibrotic areas stained in red (sections stained with Sirius Red) in 10 random fields per section from images taken at a magnification of 400×, using multiphase image analyses with ImageJ software v1.49.³⁸ (link) The surface area occupied by steatosis vacuoles was quantified in 10 random fields per section from images taken at a magnification of 200×, using multiphase image analyses with ImageJ software. Immunofluorescence was performed on 5-μm-thick cryostat sections. Sections were stained with mouse anti-CD45 (Biorbyt, San Francisco, CA, USA) antibody for 2 h at 4°C. Rabbit anti-mouse Alexa Fluor 488 (Molecular Probes) was used as secondary antibody. Hoechst 33258 dye (Sigma) was added for nuclear staining. Confocal microscopy analysis was performed using a Zeiss LSM 5 Pascal model confocal microscope (Carl Zeiss International).

Confocal microscopy analysis was performed on frozen sections for the detection of human CD133⁺ cells. Sections were labelled with the following primary antibodies: anti-CD133 mAb (1:10) (clone 293C3, Miltenyi), anti-human EPO mAb (1:200) (TermoFisher), anti-vimentin goat Ab (1:200) (Sigma) and anti-HLA rabbit Ab (1:100) (Santa Cruz Biotechnology). Rabbit anti-mouse FITC or chicken anti-goat PE or anti-rabbit PE Abs (1:500) (Molecular Probes) was used as secondary antibodies. Hoechst 33258 dye (Sigma) was added for nuclear staining.
Vessels were counted in the kidney sections stained with CD31 Ab (1:100, Santa Cruz). CD31⁺ structures were counted in non-overlapping fields (up to 10 for each section) for at least 3 mice per group using a 40× objective (HPF) in a single blind manner. Confocal microscopy analysis was performed using a Zeiss LSM 5 Pascal Model Confocal Microscope (Carl Zeiss International).

4-μm-thick cryostat sections from mouse kidney (6 per group) were incubated overnight at 4°C with an anti-CD40 rabbit polyclonal antibody (ab58391; Abcam, Cambridge, UK), or an anti-nephrin antibody (GP-N1; ProgenBiotechnic, Heidelberg, Germany; 1:100), or an anti-podocin antibody (H-130; Santa Cruz Biotechnology; 1:100), followed by the appropriate secondary antibody (Alexa Fluor 488 anti-guinea pig or anti-rabbit; Molecular Probes, Leiden, the Netherlands). Hoechst 33258 dye (Sigma) was added for nuclear staining. The number of glomeruli available on each section ranged between 5 and 10.
Confocal microscopy analysis was performed using a Zeiss LSM 5 Pascal Model Confocal Microscope (Carl Zeiss International, Jena, Germany).
Nephrin and podocin expression were analysed semi-quantitatively as previously described [35 (link)].