文章详情

Vector**组化笔H-4000使用说明书

日期：2024-05-10 02:39

浏览次数：1934

摘要：

PLEASE READ ENTIRE PROTOCOL BEFORESTARTING.

Reagents

· 2% Paraformaldehyde

· 30% Sucrose

· Superfrost™ Plus Slides (Erie Scientific Company, Cat #4951 or equivalent)

· Phosphate Buffered Saline (PBS; Sigma Chemical, Cat #P3563)

· Primary Antibody

· Wash Buffer: 0.5% bovine serum albumin, IgG-free BSA,(Jackson Immunoresearch, Cat # 001-000-162 or equivalent) inPBS.

· Blocking Buffer: 2% bovine serum albumin, IgG-free BSA,(Jackson Immunoresearch, Cat. # 001-000-162 or equivalent) inPBS.

· Qdot® Nanocrystals Secondary Antibody Conjugate

· Nuclear counter stain

· DAPI: 10 µg/ml DI water (Molecular Probes, Cat # D1306or equivalent)

Mounting media

· Gelvatol: Preparedaccording to recipe at the following link: http://www.feinberg.northwestern.edu/cif/gelvatol.htmNote: Vectashield mounting media can sometimes quench the signalfrom quantum dots. Use prepared Gelvatol or commercially availableCytoseal 60 (Richard Allen Scientific).

· ImmEdgeHydrophobic Barrier Pen (Vector Laboratories, Cat. #H-4000)

General
This protocol is intended to be astarting point for immunolabeling paraformaldehyde-fixed tissuesthat were frozen. It is expected that for a given antibody, certainsteps in this procedure will change to improve immunolabeling andreduce background staining.

Procedures

Preparing Tissue Sections:
Fix tissue in 2%Paraformaldehyde.
1. Cryo-protect in 30% sucroseovernight at 4º C.
2. Prepare 6 µm sections using acryostat and place on Superfrost™ Plus slides.
3. Keep slides at -20º C until readyfor use

Immunolabeling:
Note: All wash steps are 4minutes.
1. Rehydrate sections with 3 washesin PBS.
2. Wash 3 times with WashBuffer.
3. Block for 45 minutes at roomtemperature in Blocking Buffer.
4. Wash 5 times with WashBuffer.
5. Incubate with Primary Antibody for60 minutes at room temperature (antibody diluted in WashBuffer).
6. Wash 5 times with WashBuffer.
7. Incubate with Qdot SecondaryAntibody Conjugate for 60 minutes at room temperature (antibodydiluted in Wash Buffer).
8. Wash 5 times with WashBuffer.
9. Wash 5 times with PBS.
10. Stain with DAPI for 30 seconds atroom temperature.
11. Wash 2 times with PBS.
12. Mount coverslip with Gelvatol andrefrigerate.
13. Dry slides overnight at 4º C inthe dark.

下一篇：常用包埋剂选择指南
上一篇： Vector**组化笔H-4000美国抗体公司特别推荐！