Hoechst 33342 也称 Bisbenzimide H 33342或HOE 33342。分子式为C₂₇H₂₈N₆O·3HCl·3H₂O，分子量为615.99，CAS：23491-52-3。Hoechst 33342 是一种可以穿透细胞膜的蓝色荧光染料，对细胞的毒性较低。Hoechst 33342 染色常用于细胞凋亡检测，染色后用荧光显微镜观察或流式细胞仪检测。Hoechst 33342也常用于普通的细胞核染色，或常规的DNA染色。
Hoechst 33342 的最大激发波长为346nm，最大发射波长为460nm；Hoechst 33342和双链DNA结合后，最大激发波长为350nm，最大发射波长为461nm。本Hoechst 33342染色液可直接用于固定细胞或组织的细胞核染色，也可直接用于活细胞或组织的细胞核染色。
使用说明（参考）：
1. 对于固定的细胞或组织：
a. 对于细胞或组织样品，固定后，适当洗涤去除固定剂。随后如果需要进行免疫荧光染色，则先进行免疫荧光染色，染色完毕后再按后续步骤进行Hoechst33342染色。如果不需要进行其它染色，则直接进行后续的Hoechst 33342 染色。
b. 对于贴壁细胞或组织切片，加入少量Hoechst33342染色液，覆盖住样品即可。对于悬浮细胞，至少加入待染色样品体积3倍的染色液，混匀。室温放置3-5分钟。
c. 吸除Hoechst 33342 染色液，用TBST、PBS或生理盐水洗涤2-3次，每次3-5分钟。
d. 直接在荧光显微镜下观察或封片后荧光显微镜下观察。细胞发生凋亡时，会看到凋亡细胞的细胞核呈致密浓染，或呈碎块状致密浓染。
2. 对于活细胞或培养的组织：
a. 加入适当量Hoechst33342 染色液，必须充分覆盖住待染色的样品，通常对于六孔板一个孔需加入1ml 染色液，对于96孔板一个孔需加入100微升染色液。
b. 在适宜于细胞培养的温度培养20-30分钟。弃染色液，用PBS或培养液洗涤2-3次即可进行荧光检测。细胞发生凋亡时，在荧光显微镜下观察会看到凋亡细胞的细胞核呈致密浓染，或呈碎块状致密浓染。
注意事项：
1. 荧光染料都存在淬灭的问题，建议染色后尽量当天完成检测。活细胞或组织染色后宜立即观察。
2. 为减缓荧光淬灭可以使用抗荧光淬灭封片液。抗荧光淬灭封片液（DW2004）可以向多沃订购。
3. Hoechst 33342 对人体有害，操作时请小心，并注意有效防护以避免直接接触人体或吸入体内。
4. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品。
5. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Hoechst 33342 is also known as Bisbenzimide H 33342 or HOE 33342. Its molecular formula is C₂₇H₂₈N₆O·3HCl·3H₂O, with a molecular weight of 615.99 and CAS number 23491‑52‑3. Hoechst 33342 is a cell‑permeable blue fluorescent dye with relatively low cytotoxicity. Hoechst 33342 staining is commonly used for apoptosis detection, and samples can be examined by fluorescence microscopy or flow cytometry after staining. It is also widely applied for routine nuclear staining or general‑purpose DNA staining.
The maximum excitation wavelength of free Hoechst 33342 is 346 nm and its maximum emission wavelength is 460 nm. After binding to double‑stranded DNA, its maximum excitation wavelength shifts to 350 nm and the maximum emission wavelength is 461 nm. This Hoechst 33342 staining solution can be directly used for nuclear staining of fixed cells or tissues, as well as for nuclear staining of live cells or tissues
Instructions for Use (For Reference):
1. For fixed cells or tissues
a. After fixation of cell or tissue samples, wash appropriately to remove fixatives. If immunofluorescence staining is required, perform immunofluorescence staining first, followed by Hoechst 33342 staining. If no additional staining is needed, proceed directly to Hoechst 33342 staining.
b. For adherent cells or tissue sections, add an appropriate volume of Hoechst 33342 staining solution to fully cover the sample. For suspension cells, add at least three times the sample volume of staining solution and mix well. Incubate at room temperature for 3‑5 min.
c. Aspirate the Hoechst 33342 staining solution and wash 2‑3 times (3‑5 min each time) with TBST, PBS or normal saline.
d. Observe samples directly under a fluorescence microscope or mount sections before observation. When apoptosis occurs, the nuclei of apoptotic cells show condensed fluorescence staining or fragmented condensed staining.
2. For live cells or cultured tissues
a. Add sufficient Hoechst 33342 staining solution to fully cover the samples to be stained. Generally, 1 mL staining solution is required per well of a 6‑well plate, and 100 μL per well of a 96‑well plate.
b. Incubate for 20‑30 min at the optimal cell‑culture temperature. Discard the staining solution and wash 2‑3 times with PBS or culture medium before fluorescence detection. Under fluorescence microscopy, apoptotic nuclei exhibit condensed or fragmented condensed staining.
Precautions:
1. Photobleaching occurs with all fluorescent dyes. It is recommended to complete detection on the same day after staining. Live‑cell or live‑tissue samples should be observed immediately after staining.
2. Anti‑fade mounting medium can be used to slow down photobleaching. Anti‑fade mounting medium (DW2004) can be ordered from Duowo.
3. Hoechst 33342 is harmful to humans. Handle with care and take adequate protective measures to avoid skin contact or inhalation.
4. For scientific‑research‑use‑only by qualified personnel. Not for clinical diagnosis or therapy, and not for food‑ or pharmaceutical‑related applications.
5. Wear a laboratory coat and disposable gloves throughout the procedure for your safety and health.
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