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Biological Description
| Specificity | EAAT1 Rabbit mAb recognizes endogenous levels of total EAAT1 protein. |
|---|---|
| Background | Glutamate serves as the primary excitatory neurotransmitter in the mammalian central nervous system, but prolonged activation of its system can lead to nerve damage and cell death. After release from the presynaptic neuron and synaptic transmission, glutamate is either reabsorbed into the presynaptic neuron or neighboring glial cells via transmembrane glutamate transporters. Among these, Excitatory Amino Acid Transporter (EAAT) 1 and EAAT2 are Na+-dependent transporters predominantly expressed in glial cells of the central nervous system. They play a crucial role in regulating glutamatergic excitability by preventing excess glutamate from spilling over beyond the synaptic cleft. This function is facilitated by their ability to bind and transport glutamate into astrocytes and microglia. The activity of EAAT1 and EAAT2 is tightly regulated at multiple levels, including gene expression, post-transcriptional splicing, glycosylation, and cell-surface trafficking. While EAAT2 is more abundant and well-studied in neurotransmission, EAAT1's specific role remains somewhat unclear. However, EAAT1 expression is known to increase in response to elevated glutamate concentrations in the media of cultured primary astrocytes, suggesting potential additional significance for this glutamate transporter. Notably, EAAT1 shows neuroprotective potential following ischemic events, as reactive astrocytes and activated microglia primarily express EAAT1 rather than EAAT2 during such conditions. |
Usage Information
| Application | IF | Dilution |
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|---|---|---|---|---|---|
| Reactivity | Human, Mouse, Rat | ||||
| Source | Rabbit | MW | 58 kDa | ||
| Storage Buffer | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃ | Storage (from the date of receipt) |
–20°C (avoid freeze-thaw cycles), 2 years | ||
| IF |
Experimental Protocol:
Sample Preparation
1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.
2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.
3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.
4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.
Fixation
1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.
2. Wash the sample with PBS for 3 times, 3 minutes each time.
Blocking
Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)
Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.
Immunofluorescence Staining (Day 1)
1. Remove the blocking solution and add the diluted primary antibody.
2. Incubate the sample in a humidified chamber at 4°C overnight.
Immunofluorescence Staining (Day 2)
1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.
2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.
3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.
4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.
5. Wash with PBST for 3 times, 5 minutes each time.
Mounting
1. Mount the sample with an anti-fade mounting medium.
2. Allow the slide to dry at room temperature overnight in the dark.
3. Store the slide in a slide storage box at 4°C, protected from light.
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References
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