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银染实验

文章作者:生命科学 上传时间:2019-08-27

主干提醒:1x 40min - overnight 百分之五十 MeOH, 12% Acetic Ac1x 40min - overnight 四分之二 MeOH, 12% Acetic Acid1x 30min 八分之四 MeOH, 12% Acetic Acid, 0.05% 37% Formaldehyde3x 20min 二分之一 ETOH1x 0.2min 200 mg/l Na2S2O3 ・5H2O2x 2-3min ddH2O1x 20min 2 g/l AgNO3, 750ul/l 37% Formaldehyde1x 2-3min ddH2Ovariable 60 g/l Na2CO3, 5 mg/l Na2S2O3 ・5H2O, 500ul/l 37% Formaldehyde1x 2-3min ddH2O1x 10min 五成 MeOH, 12% Acetic Acid1x 30min �C overnight 五分之一 ETOH, 2% Glycerol

大旨提醒:1x 40min - overnight 50% MeOH, 12% Acetic Acid1x 30min&n1x 40min - overnight 二分之一 MeOH, 12% Acetic Acid1x 30min 六分之三 MeOH, 12% Acetic Acid, 0.05% 37% Formaldehyde3x 20min 贰分一 ETOH1x 0.2min 200 mg/l Na2S2O3 ・5H2O2x 2-3min ddH2O1x 20min 2 g/l AgNO3, 750ul/l 37% Formaldehyde1x 2-3min ddH2Ovariable 60 g/l Na2CO3, 5 mg/l Na2S2O3 ・5H2O, 500ul/l 37% Formaldehyde1x 2-3min ddH2O1x 10min 二分一 MeOH, 12% Acetic Acid1x 30min �C overnight 75% ETOH, 2% Glycerol

大旨提示:Coronal 30-40 µm sections cut on a freezing microtome. Sections collected into petri dishes containing 1-2ml 0.

For 1L of 0.2M phosphate buffer:6.24g of NaH2PO4.2H2O or 5.52g of NaH2PO4.H2O24.48 of Na2HPO4.2H2O or 22.72g of Na2HPO4 pH @ 7.4

  1. 0.2M acetate buffer: 3.28 g in 200 ml ddH2O. Adjust to pH6 with acetic acid.
  2. 皇家赌场号hj85,In 50 ml 0.2 M acetate buffer dissolve 2.5 g of nickel sulphate. .
  3. Add in this order: 200 mg glucose, 40 mg ammonium chloride, 50 ml DAB solution - filter into 100 ml beaker, 1.5 mg glucose oxidase

Glucose oxidase-nickel-DAB method .

  1. Coronal 30-40 µm sections cut on a freezing microtome. Sections collected into petri dishes containing 1-2ml 0.1M phosphate buffer . Sections can be kept on a shaker at 4oC for several days before commencing the immunocytochemistry. [If left too long, the sections become harder to mount].
  2. Deactivate endogenous peroxidases [For 40 ml: 20 ml 0.2M PB, 8 ml methanol, 80 µl triton-X100, 2 ml hydrogen peroxide and make up to 40 ml with ddH2O]. 15 min incubation.
  3. Wash in 0.1M PB/0.3% triton*.
  4. Preincubate in 0.1M PB/0.3% triton/1% blocking serum** for at least 1 h.
  5. Incubate at 4oC in primary antibody
  6. Wash in 0.1 M PB/0.3% triton.
  7. Secondary antibody either 2h, room temperature or overnight at 4oC @ 1:500 [made up in buffer 0.1M PB/0.3% triton/1% blocking serum].
  8. Wash in 0.1M PB. [Note: No triton].
  9. Wash once in 0.1M acetate buffer - briefly . [Acetate buffer must be made up fresh].
  10. Place sections in solution for DAB reaction . Monitor DAB reaction on microscope .
  11. Stop reaction once background is high enough by placing sections into 0.1M acetate buffer. Finally, 3 washes in 0.1M PB. Sections can be kept in cold room on share for a couple of days, or until mounted and cover-slipped.

Mount sections on gelatinised twin-frosted slides and dry in oven overnight or air dry on bench. Dehydrate with histoclear and ethanol . Coverslip with Ralmount.Use fluoromount for Fluorescent sections.

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