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PSD95 immunofluorescence detection in mouse hippocampal sections

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PSD95 immunofluorescence in prepared mouse hippocampal sections with DAPI nuclear staining.

Scope and method

Sample：Previously prepared fixed free-floating mouse hippocampal tissue sections

immunohistochemistry

Bill of materials (BOM)

StepMaterialRoleAmountRequired / optionalSubstitution conditionsSKU / specificationLiterature product
Mounting mediumGlass slides for mounting brain sectionsSupport immunofluorescence-labeled brain sections for mounting and microscopy.

Glass slides; the source does not report slide dimensions, coating or manufacturer.

Not reported in the sourceRequiredNot reported in sourceAvailable for inquiryNot reported in source
Coverslip sealingCoverslips for mounted brain sectionsCover mounted brain sections; seal the edges using the nail polish specified in the protocol.

Coverslips used with the slides and mounting step; the source does not report dimensions or thickness.

Not reported in the sourceRequiredNot reported in sourceAvailable for inquiryNot reported in source
Coverslip sealingSeal the edges of the coverslip with nail polishCoverslip edge mounting

Mixture suitable for mounting the edges of coverslips

Cover the edge of the coverslipRequiredNot reported in sourceAvailable for inquiryNot reported in source
Section washingTBST Tissue Section Wash Buffer (TBS, 0.2% Triton X-100)Brain slice washing

TBS and Triton X-100 mixed working solution

1× TBS；0.2% Triton X-100RequiredNot reported in sourceAvailable for inquiryNot reported in source
Fluorescent secondary antibody bindingAlexa Fluor-conjugated secondary antibody working solution (1:200, PSD95 rabbit primary antibody channel)Fluorescence readout

Alexa Fluor-conjugated secondary antibody, paired with the selected rabbit-derived PSD95 primary antibody

1:200RequiredNot reported in sourceAvailable for inquiryNot reported in source
Mounting mediumBrain section glycerol/Tris/n-propyl gallate mounting mediumSection mounting

Multi-component mounting medium prepared according to the source formulation

90% glycerol; 20 mM Tris, pH 8.0; 0.5% n-propyl gallateRequiredNot reported in sourceAvailable for inquiryNot reported in source
DAPI nuclear stainingDAPI Nuclear Staining Working Solution (1:50000, immunofluorescence of brain sections)Nuclear counterstaining

DAPI nuclear staining working solution, to be used in conjunction with the fluorescence detection system employed

1:50000RequiredNot reported in sourceAvailable for inquiryNot reported in source
Serum blocking10% normal goat serum/TBST blocking and primary antibody dilution bufferBlocking and primary antibody dilution

TBST working solution prepared with normal goat serum

10%RequiredNot reported in sourceAvailable for inquiryNot reported in source
PSD95 primary antibodyPSD95 Rabbit Antibody (51-6900)PSD95 detection primary antibody

Rabbit IgG polyclonal, PSD95 target, catalog number 51-6900; secondary antibody paired with rabbit-derived primary antibody.

1:300RequiredNot reported in sourceAvailable for inquiryNot reported in source
Inquire about the whole BOM

Bulk pricing and availability inquiry

Submitting an inquiry does not place an order or confirm a purchase.

Protocol #184 · revision 3 Glass slides for mounting brain sections | Not reported in the source | Available for inquiry Coverslips for mounted brain sections | Not reported in the source | Available for inquiry Seal the edges of the coverslip with nail polish | Cover the edge of the coverslip | Available for inquiry TBST Tissue Section Wash Buffer (TBS, 0.2% Triton X-100) | 1× TBS；0.2% Triton X-100 | Available for inquiry Alexa Fluor-conjugated secondary antibody working solution (1:200, PSD95 rabbit primary antibody channel) | 1:200 | Available for inquiry Brain section glycerol/Tris/n-propyl gallate mounting medium | 90% glycerol; 20 mM Tris, pH 8.0; 0.5% n-propyl gallate | Available for inquiry DAPI Nuclear Staining Working Solution (1:50000, immunofluorescence of brain sections) | 1:50000 | Available for inquiry 10% normal goat serum/TBST blocking and primary antibody dilution buffer | 10% | Available for inquiry PSD95 Rabbit Antibody (51-6900) | 1:300 | Available for inquiry

Procedure

  1. Section preparation

    Use previously prepared fixed free-floating mouse brain tissue sections.

  2. Section washing

    Wash brain sections in 1× TBS containing 0.2% Triton X-100 (TBST).

  3. Blocking

    Block nonspecific binding.

  4. Serum blocking

    Incubate sections in 10% normal goat serum prepared in TBST for 1 h at room temperature.

  5. Primary antibody incubation

    Immunolabel with a rabbit primary antibody against PSD95.

  6. Primary antibody binding

    Dilute the primary antibody in 10% normal goat serum/TBST and incubate at 4°C with gentle shaking for 2–3 nights.

  7. PSD95 primary antibody

    Rabbit antibody against PSD95, catalog number 51-6900, diluted 1:300.

  8. Secondary antibody incubation

    Use an Alexa Fluor-conjugated secondary antibody matched to the rabbit primary antibody.

  9. Washing after primary antibody incubation

    Wash sections with TBST.

  10. Fluorescent secondary antibody binding

    Dilute the Alexa Fluor-conjugated secondary antibody 1:200 in TBST and incubate at room temperature for 2–3 h.

  11. Mounting

    Mount the sections after completing immunolabeling.

  12. Washing before mounting

    Wash sections with TBST.

  13. Mounting medium

    Mount sections on slides using a mounting medium prepared from 90% glycerol, 20 mM Tris (pH 8.0), and 0.5% n-propyl gallate.

  14. Coverslip sealing

    Seal the edges of the coverslip with nail polish.

  15. Nuclear labeling

    Stain nuclei with DAPI.

  16. DAPI nuclear staining

    During the final 10 min of secondary antibody incubation, add DAPI to the secondary antibody solution at 1:50,000.

  17. Fluorescence imaging

    Record fluorescence images of immunolabeling and nuclear staining.

  18. Image acquisition

    Acquire images using a fluorescence microscope, for example an Olympus FV 3000.

Quality control and readouts

Use a secondary antibody matched to the rabbit primary antibody; record PSD95 fluorescence and DAPI nuclear fluorescence separately.

PSD95 immunoreactivity and its spatial distribution in hippocampal sections, using DAPI-labeled nuclei as localization references.

Study design
Compare PSD95 immunoreactivity in hippocampal sections from four groups: wild-type–vehicle, wild-type–NRG1, Tg2576–vehicle, and Tg2576–NRG1.

References

  1. PSD95 immunofluorescence detection in mouse hippocampal sections · DOI: 10.17504/protocols.io.j8nlk8m4xl5r/v2
  2. Ryu J, Hong B-H, Kim Y-J et al. Neuregulin-1 attenuates cognitive function impairments in a transgenic mouse model of Alzheimer's disease. 2016. DOI:10.1038/cddis.2016.30; PMID:26913607. · DOI: 10.1038/cddis.2016.30
  3. Thermo Fisher Scientific / Invitrogen. PSD-95 Polyclonal Antibody, catalog 51-6900, RRID:AB_2533914.

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