Quantification of Immunostained Caspase-9 in Retinal Tissue

This protocol provides a detailed immunohistochemical method to identify, validate, and target functionally relevant caspases in complex tissues. The main advantage of this technique is that enables the interrogation of cell type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

Begin quantifying the caspase levels using confocal microscopy images of stained retinal sections. To do so, drag the image files of 405, 470, 555 and 640 channels to Fiji's console. Click image, color, and merge channels tabs to assign colors to the files.

Then compress the Z stack by clicking the image stack and Z project. Select the projection type as max intensity. Open the channel's interface by clicking the image followed by color channel's tool.

Next, open the brightness and contrast interface. To select the parameters per channel, go to the channel's tab and choose one channel. Put the cursor on top of the background and annotate the pixel value.

Then put the cursor on the caspase expressed cell and annotate the pixel value. To test the parameters, go to the brightness and contrast interface and click select. Plug in the minimum displayed value or the caspase expressed cell's pixel value.

Then plug in the maximum displayed value or the background pixel value before clicking okay. Choose the random images from blinded tissue and repeat the selection of parameters per channel to test the parameters for all the channels. Once the parameters are set, open the image files, and compress the Z stack.

Add the brightness and contrast parameters for the caspase channel and the isolectin for the vascular marker channel. Use the point tool and quantify the number of positive vascular areas using colocalization of the vascular marker with caspase expression as the readout of positive areas. Then using hooks as the marker of positive neuronal areas, quantify the number of positive vascular areas.

Annotate the values on a spreadsheet per image and average the values by section. Average the section values, which will be the readout per eye. The protocol identified the cellular localization of the caspases and the neuronal retinal layers in which the caspases are expressed.

The retinal cross section allowed the visualization of retinal nuclei in the retinal ganglion layer or RGL, Internuclear layer or INL, and the outer nuclear layer or ONL when stained with hooks. In the cross section, the retinal blood vessels appeared disconnected and separate supplying the plexiform layers between the RGL and INL and between the INL and ONL. Retinal cross sections also identified the highly regulated caspase-9, one day post retinal vein occlusion.

The caspase-9 inhibition was more effective at reducing neuronal caspase-7 expression in endothelial cell knockout mice which validated the functional relevance of endothelial caspase-9. The representative analysis showed that the inhibition of caspase-9 activity pharmacologically blocked the caspase-7 expression post retinal vein occlusion. The success of the technique relies on careful tissue preparation as well as IHC and microscopic imaging to ensure consistency, validity, and reliability of the data.

This method could be combined with western blotting analysis for semi-quantitative comparison of caspase signaling. The technique provided the steps to interrogate the efficacy of a therapeutic approach targeting active caspase-9 in RVO. This approach can also be applied to other tissues, including the brain.