生物化学技术

DAY ONEParaffin sections:1) Deparaffinize: Xylene , 3x, 5 min each2) Rehydrate: 100% EtOH, 2x, 5 min each95% EtOH, 2x, 5 min each80% EtOH, 1x, 5 min50% EtOH, 1x, 5 minProceed to Antigen Retrieval below (step 3)Cryo sections (starting with slides that have been baked @ 50°C, 30min):1) Fix in 4% PFA/PBS, 15min2) rinse wi ...

Double immunohistochemistry for BCL6 and PNA for Germinal Center detection.(on fixed, paraffin-embedded sections)Cut sections at 4µm, use a clean water bath with distilled water, and let the sections dry upright in order to facilitate adhesion between the section and the charged glass s ...

Solutions100X trypsin10 mg/ml in 1X PBSSigma #T2271100X soybean trypsin inhibitor10 mg/ml in 1X PBSSigma #T65221000X DNaseI2mg/ml in HBSSSigma #D4513Explant Culture Medium45% HAMS F-12 (Gibco #11765-054)45% DME (Gibco #10313-021)10% FCS (HyClone)Insulin (5 mg/ml 1000X sto ...

Staining mouse tissue with anti-mouse Ig (H&L chain) secondary reagents inevitabily causes staining of interstitial immunoglobulins, B cells, plasma cells and macrophages with Ig bound to Fc receptors. To avoid this, staining with F(ab) monomeric secondary has been developed (Refer ...

Double ImmunohistochemistryPreface:You should not co-stain in IHC antigens located on the same structure (nucleus, membrane, cytoplasm). That because the success of this double staining procedure is based on the complete development of the first stain, which should mask the struct ...

Immunohistochemistry on fixed, paraffin-embedded sections.Rationale: Immunostaining on formalin-fixed, paraffin embedded sections has been revolutionized in 1991 by the discovery of heat-mediated retrieval (Antigen Retrieval, AR) of immunoreactivity (Ref. 1, 2 ). This m ...

Any type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved. Use cytocentrifuged cells or frozen or paraffin dewaxed sections. Presence of endogenous fluorescent substances may affect your staining. Note: This protocol uses a twice repea ...

Double staining for Ag and Ki-67, TUNEL or BCL-6.Cut frozen tissue sections (spleen, lymph nodes) in a cryostat at ~ 4µm, on plain glass slides (Adhesive -coated slides are sometimes too hydrophobic, but fine if you have them). Let them dry approx 1 hour in a dry atmosphere at RT. Antibodies (anti-Pe) in the sect ...

PurposeMaterials10ml 6% dextran + 7ml citrate/citric acid Dextran: T500 -- 6g+100ml PBS Citrate solution: 25g Na Citrate + 8g citric acid + 500 ml PBS 43 ml blood 12 ml RT Histopaque 1077 18 ml cold H2O 2 ml 10x PBS M199 for HUVECs: 1L powder pocket M199 + 35g NaHCO3 + 25mM Hepes + 5 mM glutamine + 50 ug/ml Gentamycin. 1M Hepes: 11 ...

Human ELISPOT ProtocolHuman ELISPOT Protocol（TNFa,IL13, IL12,IL10, IL6,IL5,IL4,IL2,IL1b,IFNg,FasL） the protocol is in the file below: click here to download 上一篇：peritoneal macrophage preparation 下一篇：ELISPOT Assays Visualize the Secretory Product of Individual Cells

ELISPOT 技术简介　　随着酶联免疫分析技术在医学及生物学领域的广泛应用 , 使体外检测各种细胞因子及抗体研究有了新的突破。在研究免疫应答机制时以往常用用酶联免疫吸附法（ ELISA ）检测体液中游离的细胞因子（ CK ）或抗体，但由于游离的循环抗体或 CK 的半哀期不同，使之在体液中不断的被代谢或与靶器官结合，而不能确切的反映体内的抗体及 CK 的水平。 80 年代，国外的科研工作者根据 ELISA 技术的基本原理，建立了体外检测特异性抗体分泌细胞和 CK 分泌细胞的固相 ...

ELISPOT Assays Visualize the Secretory Product of Individual CellsELISPOT assays are exceptionally sensitive since the product is captured directly around the secreting cell, before it is either diluted in the supernatant, captured by receptors of adjacent cells, or degraded. This ...

ELISpot assays employ the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Either a monoclonal or polyclonal antibody specific for the chosen analyte is pre-coated onto a PVDF (polyvinylidene difluoride) -backed microplate. Appropriately stimulated cells a ...

简介ELISPOT技术　　ELISPOT全名为Enzyme-linked Immunospot Assay，其技术原理与ELISA相似。其实验设计是在96微孔培养盘底部披覆PVDF薄膜，用来吸附特殊挑选、且无毒性（不含sodium azide、内毒素endotoxin）的单株抗体。静脉血PBMC细胞经适当分离处理后，会被分配到微孔盘上，再接受适当的抗原刺激，并将微孔盘放置于温箱中过夜培养一段时间。一般而言，记忆型T细胞在受抗原刺 ...

Protocol: Immunofluorescence / confocal microscopyB or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections of unfixed, OCT embedded tissue:1. wash cells 1x cold RPMI (no wash for cryostat sections).2. Fix 20 min 4% paraformaldehyde in 0.1M p ...

Materials: 1) 1.0M KPO4 pH 6.5 : Make by mixing 33 mls of 1M K2HPO4 with 67 mls of 1M KH2PO4 2) 0.1M KPO4 pH 6.5 3) 0.1M KPO4 pH 7.5: Make 500 mls by mixing 41.7 mls of 1M K2HPO4 and 8.3 mls of 1M KH2PO4 with 450 mls of ddH2O 4) 37% Formaldehyde 5) KS Buffer: 0.1 M KPO4 pH 7.5/1.2 M Sorbitol 6) Zymolyase 100T: 5 mg/ml solution in 0.1M KPO4/0.5% β-mercaptoethan ...

Reagents: NGS-Normal Goat Serum PBST-0.5% Tween-20/CMF-PBS CMF-PBS 1°Ab-mouse MF20 (anti-myosin monoclonal) 1°Ab-rabbit myoD (anti-myoD polyclonal) 2°Ab-goat anti-mouse IgG-FITC 2°Ab-goat anti-rabbit IgG-FITC Procedure: You will be staining one of your transfected chambers ...

《细胞培养》 司徒镇强主编，2004【步骤】（以间接免疫荧光法为例）1 细胞准备与固定 （1）单层生长细胞：取对数生长细胞，用0.25％胰蛋白酶消化，制成单细胞悬液。将细胞接种到预先放置几张6×22mm盖玻片的培养瓶或培养皿中，置二氧化碳培养箱培养1-3天，待细胞接近长成单层，取出盖玻片，进入PBS（0.01 mol/L,pH7.4）洗2次，然后根据实验目的，选择适当固定剂固定细胞。常用固定剂有：95％乙醇（固定时间10-30min），丙酮（5－1 ...

Spin 2 ODU of cells 3000rpm, 5'Resuspend in 5ml of 0.1M KPi pH 7.0Add 0.6ml 37% formaldehyde and rotate (on nutator) at r.t. for 2 hrs.(can store cells fixed at 4'C O/N)Spin down cells (2500 rpm, 2')Wash 2x in 5ml 0.1M KPi pH 7.0Wash 1x in 5ml 1.2M Sorbitol in 0.1M KPi pH 7.0Resuspend in 1ml 1.2M Sorbitol/0.1M KPi pH 7Add 5ul B-mercapt ...

Note :this protocol is a modified version of the protocol from Mark Rose in the CSH Yeast Genetics Course Manual.Grow cells at the appropriate temperature to 5x10E6 in 5 mls YPD. Add 0.5mls 37% formaldehyde (best grade) and incubate on the roller at same temp. for 10 min. Spin down cells 2Kx3min. and resuspend ...