Immunohistochemical staining of 7 μm wax sections of full thickness retina were performed. Sections from wax blocks from each group (n = 4 retinas/group) were used for immunohistochemical analysis. To prevent non-specific background, tissues were incubated in 1% bovine serum albumin (BSA, Sigma)/PBS for 1 h at room temperature before incubation with the primary antibodies for 16 h at 4 °C. After several wash steps, the tissues were incubated with the secondary antibodies (sTable 1 ) for 4 h at 4 °C and subsequently washed with PBS. The sections were counterstained with the nucleic acid stain Hoechst 33342 (1 μg/ml; Invitrogen) in PBS. Images were acquired with Olympus FluoView1000 confocal microscopy (Olympus, Tokyo, Japan) or Leica SPE-II confocal microscope (Leica, Wetzlar, Germany). Each target protein fluorescent integrated intensity in pixel per area was measured using the ImageJ software. All imaging parameters remained the same and were corrected with the background subtraction.
Leica spe 2 confocal microscope
Manufactured by Leica camera
Sourced in Japan
The Leica SPE-II confocal microscope is a high-performance imaging system that uses laser technology to capture detailed and high-resolution images of microscopic samples. It is designed to provide optical sectioning and improved contrast, enabling users to visualize intricate structures and features within their specimens.
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2 protocols using leica spe 2 confocal microscope
1
Immunohistochemical Analysis of Retinal Tissue
2
Biofilm Quantification Using Confocal Microscopy
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Biofilms on coupons were stained by adding 10 μL of PI (100 μM/L) and SYTO 9 (100 μM/mL) on their surface and incubated in the dark for 5 min. The samples were gently covered with a coverslip and were observed at room temperature using a Leica SPE-II confocal microscope (Leica) equipped with solid-state lasers for excitation. Images were acquired under × 60 magnification oil immersion objective lens with a Leica DFC500 camera and Leica LAS AF software at a 1-μm interval through the biofilms and five image stacks, each representing a different field of view on the coupon. Samples were excited using a 488 nm laser and fluorescence was detected at 617 nm (PI) and 503 nm (SYTO 9). The thickness (μm) of biofilms was measured using the Leica LAS AF software. The confocal images were Z-stacks of optical sections using 512 × 512-pixel resolution tagged image file format. From the Z-stacks of the 3D biofilm structure the biomass volume (μm3 / μm2) was calculated using COMSTAT software (Heydorn et al. 2000 (link); Vorregaard 2008 ) and the percentage of live/dead cells was determined.
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