mouse retinal tissue fixation and immunostaining for pre&#45;embedding immunoelectron microscopy

Synaptic Circuits Analysis and Protein Localization in the Mouse Retina

The complexity of neuronal circuits in the retina presents challenges for exploration, considering the diverse cell types within its structure1,2. The initial step involves identifying synaptic connections between different cells and determining the cellular localization of specific neurotransmitters or neuropeptides. As molecular biology advances introduce new proteins, precise localization in the retina becomes crucial for understanding their functions and analyzing retinal circuits and synaptic connections3,4,5.
Due to the limited resolution of light microscopy, electron microscopy (EM) is commonly used to detect the subcellular structures of nerve cells. EM has various classifications, with conventional transmission electron microscopy (TEM) utilized for observing cell ultrastructures6,7,8,9. Immunoelectron microscopy (immuno-EM), which combines the spatial resolution of EM with the chemical identification ability of antibodies binding specifically to proteins10, stands out as the optimal and exclusive method for investigating synaptic connections and subcellular protein localization in the retina11,12.
Immuno-EM techniques can be divided into pre-embedding and post-embedding methods based on the order of embedding and antibody incubation. Compared with the post-embedding method, the pre-embedding approach is capable of large-scale and long-distance identification13,14,15, offering an optimal approach for studying cell processes like axons and dendrites. Additionally, this technique provides a strong signal and broad field of view, making it advantageous for comprehensive investigations of protein expression and molecular localization in the cytoplasm. This method proves particularly valuable in ensuring chemically identified structures that are visible throughout the entire cytoplasm, cells, or retina.
However, the post-embedding method, while having lower penetration or diffusion compared to the pre-embedding method, is not as sensitive16,17. In simple terms, if the goal is to explore the localization of specific neurotransmitters in the cytoplasm or synaptic terminals, the pre-embedding immuno-EM is the preferred method. Conversely, for identifying the localization of membrane receptors, it is more recommended to utilize post-embedding immunogold EM.
Given these considerations, we opt for the pre-embedding immuno-EM method to delve into retinal circuits, including the interaction between cone and rod pathways, and molecular localization, such as the distribution and synaptic organization of substance P-like immunoreactivity (SP-IR) in the mouse retina.

Author Spotlight: Understanding the Ultrastructural Basis of Retinal Synaptic Connectivity and Neurotransmitter Localization in Mice 

Investigating Retinal Circuits and Molecular Localization by Pre&#45;Embedding Immunoelectron Microscopy

In our research, we focus on retinal physiology and the disease. They need to investigate the cellular composition and the specific neurotransmitters within the retina neural circuit. The overarching goal is to explore synaptic connections and the cellular localization of neurotransmitters throughout the entire pathway.The biggest challenge of immuno electron microscope technology is difficult in achieving a balance between structure, integrity, and positive signals. Through body immunoelectron microscopy, we provide for the first time immunochemically verified ultrastructural evidence for the labs and the review and explicit distribution, novel organic cell features, and the synaptic organization or SPIR is the most All findings suggest that the crosstalk between and the raw pathway is much more extensive than previously assumed. This neurochemically identified ultrastructural results reveal that on and off signaling may interconnect through chemical synapse in the retina's OPL.

Mouse Retinal Tissue Fixation and Immunostaining for Pre&#45;Embedding Immunoelectron Microscopy

To begin, assemble a dissecting microscope, two forceps with fine tips, scissors, a one milliliter syringe needle, and filter paper on a working platform. Next, using elbow scissors, nucleate the eyes from an anesthetized mouse. Place the eyes into a glass dish containing 4%paraformaldehyde and 0.2%picric acid in 0.1 molar phosphate buffer.On the dissecting microscope, using a one milliliter syringe, punch a hole at the corneal limbus and cut off the anterior segment with scissors. Use forceps to remove the lens from the inner retinal surface. Now using two forceps, carefully peel the sclera until the retina is completely isolated from the eye cup.Subsequently, cut the retina into four pieces. After overnight fixation and 4%paraformaldehyde solution, wash the retinal tissues in 0.01 molar PBS six times for 10 minutes each. Incubate the retinal tissue in 1%sodium borohydride in 0.01 molar PBS for 30 minutes.Again, wash the retinal sections in 0.01 molar PBS at least six times. Then, remove the vitreous with filter paper. And using a double-edged razor blade, slice the retina into small sections between 100 and 300 micrometers.Wash the retinal stripes in 0.01 molar PBS six times for 10 minutes each before incubating them with Reagent A and Reagent B from the ABC kit for two days. Next, wash the retinal stripes in 0.05 molar tris buffer three times for 10 minutes each. Pre-incubate the retinal stripes with 5%Reagent 1 and 5%Reagent 3 from the diaminobenzene, or DAB kit, in distilled water for one hour at room temperature.To stain the retinal stripes with DAB, add the same volume of solution from three tubes in the DAB kit in sequence. Under the dissection microscope, observe the staining condition. Wash the retinal strips with 0.05 molar tris buffer three times for 10 minutes each.Finally, wash the strips in 0.01 molar PBS six times for 10 minutes each. In control experiments, cone pedicles with two ribbons and rod spherical with one ribbon showed no immunoreactivity in the absence of primary antibodies. Protein kinase C alpha identified rod bipolar cells in the retina.The DAB standing showcases the presence of protein kinase C alpha and bipolar cell dendrites, forming synapses with rod and cone terminals in the outer plexiform layer. Additionally, the DAB reaction product aids in identifying rod bipolar cell terminals and axonal processes in the inner plexiform layer. Substance P immunoreactivity amacrine cells were shown to be presynaptic to substance P negative amacrine cells.Furthermore, substance P immunoreactivity amacrine cells were post-synaptic to bipolar terminals in the sublayer three and sublayer five levels of the inner plexiform layer, respectively.

mouse-retinal-tissue-fixation-immunostaining-for-preembedding

Research

JoVE Journal

Neuroscience

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

This protocol outlines the detailed steps of pre-embedding immunoelectron microscopy, with a focus on exploring synaptic circuits and protein localization in the retina.