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HOW TO STAIN PFA-FIXED BRAIN TISSUE USING GOLGI-COX METHOD

Golgi–Cox method requires impregnation of fresh unfixed brain blocks. However, if PFA-fixed or formaldehyde-fixed tissues have to be used, some special treatments for fixed tissues should be performed to get good results.

1. The animal must be completely perfused with buffer and fixative. Perfusion should be performed within five minutes after death of the animal. Once tissues are removed, transfer them into the fixative as soon as possible. Fixed and sucrose solution treated tissues can be stored at -70°C for a long time.

2. Before Golgi-Cox staining, the fixed tissue needs to be placed under running tap water for at least 24 hours.

3. Transfer tissue into distilled water and store at room temperature in the dark for another 24 hours.

4. Transfer tissue into the impregnation solution and follow Tissue Preparation protocol from step 4 like unfixed Golgi-Cox brain tissues (Hito Golgi-Cox OptimStain™ Kit User Manual Page 6 – Standard Protocol or Page 8 – Vibratome Protocol) After the treatment, staining quality with fixed tissue is satisfactory for demonstrating dendritic branching pattern and dendritic spines. Glia cells can also be stained. Compared to the fresh tissues, PFA-fixed tissues or formaldehyde-fixed tissues have stained glia cells which contribute to higher background. Though we do not recommend using fixed tissues, our HITO GOLGI-COX OPTIMSTAIN™ KIT provides an alternative approach to do research with fixed pathological samples.

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How to make gelatin coated slides for Golgi staining?

Gelatin coated slides can hold Golgi sections on slides and prevent sections falling off. Using Hito Gel-Coat™ Solution for Golgi Staining, everyone can make low-cost gelatin coated slides with high quality like commercial grade products.
Procedure: Heat the water bath or oven to 50-60 °C, pre-warm the HITO GEL-COAT™ SOLUTION FOR GOLGI STAINING and allow the solution to completely liquefy, using a 50ml Coplin jar, dip slides (pre-cleaned) in the warm solution three times, drain the slides onto paper tissue, and then carefully stand the slides on end to air dry or in incubator at 37°C overnight. Store slides in slides box at room temperature.

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RESEARCH TOPIC- GOLGI STAINING PARAFFIN SECTION – RESEARCHGATE

RESEARCH TOPIC- GOLGI STAINING PARAFFIN SECTION – RESEARCHGATE
Link for a Research Topic – ResearchGate, our HITO GOLGI-COX OPTIMSTAIN™ KIT will be a solution

Is it preferable to do golgicox staining for brain in paraffin embedded tissue section?

https://www.researchgate.net/post/Is_it_preferable_to_do_golgicox_staining_for_brain_in_paraffin_embedded_tissue_section

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HOW TO PREPARE GOLGI STAINING SAMPLES USING PARAFFIN EMBEDDING METHOD

HOW TO PREPARE GOLGI STAINING SAMPLES USING PARAFFIN EMBEDDING METHOD

JANUARY 2, 2017 11:53:00 AM EST

How to prepare golgi staining samples using paraffin embedding method

  1. Prepare Hito Golgi-Cox OptimStain™ Kit Impregnation Solution
  2. Prepare animal for infusion by administering a lethal dose of anesthesia. Monitor it until the point when the animal fails to respond to pinching of the foot.
  3. Do not perfuse with buffer or fixative. Remove the tissue (brain, spinal cord or heart) as soon as possible.  Handle with care and avoid damage of the tissue. Large specimens should be sliced with a sharp blade into blocks of approximately 10 mm thickness.
  4. Rinse tissue in double distilled water for 2-3 seconds to remove blood from the surface.
  5. Transfer tissue into the mixed impregnation solution that is at least five times the volume of the tissue and store at room temperature in the dark.
  6. Replace the impregnation solution on next day (after 12-24 hours), and store at room temperature (20 – 25°C) for two weeks in the dark. To avoid non-specific staining, do not extend the impregnation time.
  7. Transfer tissue into Solution-3 that is at least five times the volume of the tissue. Store at 4°C in the dark. Replace Solution-3 after 24 hours, and continue to store at 4°C in the dark for 2-3 days.
  8. After 2-3 days processing in Solution-3, transfer the tissue into a new vial and wash the tissue with double distilled water overnight at room temperature.
  9. After water rinse, dehydrate the tissue in a graded ethanol/water series at room temperature. Clear tissue in chloroform for 12-24 hours. Process tissues in paraffin for 2-4 hours, preferably in a vacuum oven. Embed tissues in paraffin blocks (recommend paraffin with a lower melting point ). Note: Xylene can also be used as the clearing agent. The tissues cleared by chloroform will be softer and thus easier to cut. Using xylene as the clearing agent sometimes can lead to cracks of tissue sections.
  10. Turn on the water bath and set the temperature at 45-50°C. Use fresh deionized water. Insert the paraffin block into the microtome chuck. Set the dial to cut 50-80 µm sections. Cut sections very slowly and pick them up with forceps or a fine paint brush and float them on the surface of the water bath. Float the sections onto the surface of HITO DUAL-SAFE™ GELATIN-COATED SLIDE.
  11. Drain slides upright and dry at 37°C for a minimum of 15 minutes.
  12. Place the slides with paraffin sections in a 60°C oven for 2 hours to bond the tissue to the glass. Slides can be stored in a slide box at room temperature for three months.
  13. Deparaffinize sections in xylene, 3 times, 5 minutes each.
  14. Rehydrate sections in 100%, 95%, 70% and 50% ethanol 2 times, 5 minutes each.
  15. Rinse slides in distilled water 3 times, 3 minutes each.
  16. Mix 2 ml Solution-4, 2 ml Solution-5 and 6 ml double distilled water in a 12 ml staining jar (provided in the kit), then place slides in the solution mixture. Tightly close the staining jar and wait for 10 minutes.
  17. Rinse slides in distilled water 2 times, 4 minutes each (distilled water should be renewed frequently).
  18. Counterstain sections with cresyl violet (optional step).
  19. Dehydrate slides which was previously rinsed with distilled water, in 50%, 75% and 95% ethanol, 5 minutes each.
  20. Dehydrate slides in 100% ethanol, 3 times, 5 minutes each.
  21. Clear in xylene, 2 times, 5 minutes each, and apply coverslip over sections using undiluted xylene based resinous mounting medium.
  22. Allow to dry. The slides can be viewed after drying by bright field microscopy.

paraffin section / golgi cox staining

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Hito Golgi-Cox OptimStain™ Kit User’s Guide. & Safety Manual (Chinese translation)

HITO 高尔基优化染色试剂盒简要使用说明

Hito Golgi-Cox OptimStain™ Kit User’s Guide. & Safety Manual (Chinese translation)

From Dr. **  ***, ** Hospital, BeiJing, China (Author personal information is removed)

警告: 此试剂盒包含有毒试剂,应在通风橱下进行实验,并同时使用防护服,手套,安全眼镜等实验室常规防护措施。操作结束后,用肥皂和水彻底清洗双手。

如误食:用水漱口,并立即就医。如果接触到皮肤或眼睛:立即用大量的水清洗并就医。

绝对不要将废液倒入下水道,应将废液收集在密闭的玻璃或高密度聚乙烯容器中并由专业的废物处理人员去处理。欲了解更多信息,请阅读产品安全数据说明MSDS。

I. 溶液-1 准备

Hito 高尔基优化染色试剂盒中,溶液-1含有有害的试剂。根据相关法律,为了保证运输和使用时的安全性,溶液-1被设计为两部分:浓缩液及准备液。含有毒试剂的浓缩液依据美国交通部有害物包装运输最高标准独立包装。

收到试剂盒后,小心地将溶液-1A(浓缩液)倒入溶液-1B(准备液),混匀后溶液-1的最终体积为250ml(标准试剂盒)或125毫升(小试剂盒)。

标记溶液-1瓶标签上的浓缩液(A)标记框,以注明瓶内含有有毒试剂。

此混合溶液为完整的溶液-1。

II. 浸泡液制备

清洁所有的容器并用蒸馏水冲洗。避免使用金属工具。在一个干净的有盖玻璃或塑料容器中混合等体积的溶液-1和-2,盖紧容器,室温下静置避光(例如用铝箔包裹)贮存至少24小时后方可使用,此混合液应在一个月内用完。

浸泡组织应使用此混合溶液的上清液(无沉淀物)。浸泡液的量应为被浸泡组织的至少五倍,(1立方厘米的组织使用5毫升以上的浸泡液)。

IIIA. 组织准备 A(使用冰冻切片机的标准解决方案)

  1. 深度麻醉动物。不要使用缓冲液或固定液灌注。小心快速取出组织(脑,脊髓)。大型标本应用锋利的刀片切成厚度约10毫米的组织块。双蒸水冲洗组织2-3秒,以除去表面的血液。
  2. 将组织转移到至少5倍体积的制备好的浸泡液中,并在室温下避光贮存。
  3. 第二天(12-24小时)更换浸泡液,室温下(20 – 25°C)避光浸泡两周。为了避免非特异性染色,不应延长浸泡时间。
  4. 两周后将组织转移到至少5倍体积的溶液-3中,避光贮存于4℃,12小时后更换溶液-3,在4℃下继续避光贮存24〜72小时,从溶液-3取出组织冷冻前,应使用滤纸小心去除多余的溶液-3。
  5. 在一个金属容器或玻璃烧杯中加入300〜500毫升异戊烷后放置放置于干冰中15〜30分钟,直到异戊烷温度降至-50℃〜-70℃。
  6. 将组织或包含组织及OCT的包埋模具小心放入金属网篮(可用铝箔自制),慢慢浸入冷却的异戊烷,持续30秒到1分钟。 (浸渍冷冻的时间非常关键,既要保证组织完全冻结,又要避免组织爆裂,必要情况下,应根据不同实验条件测试不同的时间长度,以确定最佳的时长)。
  7. 冷冻完成后,使用预冷的镊子迅速取出冰冻组织,并将其放置在干冰盒中的滤纸上以去除多余的异戊烷。
  8. 将冰冻组织用铝箔包裹并储存在-70℃下,等待切片。
    冷冻组织可以使用下列简化程序:

使用滤纸小心去除多余的溶液-3后,将组织置于铝箔上或置于灌入OCT的模具中,直接放置于 -70℃或-80℃冰箱中15到30分钟。冷冻完成后,将冰冻组织用铝箔包裹并储存在 -70℃下,等待切片。

  1. 将冰冻切片机温度设定在-19°C,使用OCT或蒸馏水将组织冻结在切片机样品座上并固定在切片机样品头上,设定切片厚度为80-200微米即可切片(-19℃温度设定在大多数情况下是令人满意的,但需要根据不同的切片机和组织类型加以优化)。
  2. 使用滴瓶在涂布明胶的载玻片上滴加少量溶液-3,用附带的毛笔涂开,将切片使用附带的毛笔或玻璃棒移到载玻片上,用滤纸条除去过量的溶液-3,在室温下斜立避光过夜晾干。干燥的切片应尽快进行下一步处理,如情况不允许,也可以在室温下避光存储二到三天。但切不可长时间存放,否则将导致黑色的晶体状背景。
    IIIB. 组织准备B(使用振动切片机的解决方案)
  3. 深度麻醉动物。不要使用缓冲液或固定液灌注动物。小心快速取出组织(脑,脊髓)。大型标本应用锋利的刀片切成厚度约10毫米的组织块。双蒸水冲洗组织2-3秒,以除去表面的血液。
  4. 将组织转移到至少5倍体积的制备好的浸泡液中,并在室温下避光贮存。
  5. 第二天(12-24小时)更换浸泡液,室温下(20 – 25°C)避光浸泡两周。为了避免非特异性染色,不应延长浸泡时间。
  6. 两周后将组织转移到至少5倍体积的溶液-3中,避光贮存于4℃,12小时后更换溶液-3,在4℃下继续避光贮存24〜72小时。
  7. 使用低温琼脂糖凝胶包埋组织,用刀片修块后,将样本固定在振动切片机样品座上,设定切片厚度为80-200微米即可切片,将切片收集到双蒸水中,使用附带的毛笔将悬浮在蒸馏水中的切片贴到涂布明胶的载玻片上,用滤纸条除去过量的双蒸水后,使用滴瓶在组织切片上滴加少量溶液-3,等待1-2分钟后,用滤纸条除去过量的溶液-3,在室温下斜立避光过夜晾干。干燥的切片应尽快进行下一步处理,如情况不允许,也可以在室温下避光存储二到三天。但切不可长时间存放,否则将导致黑色的晶体状背景。可长时间存放,否则将导致黑色的晶体状背景。
    IV. 染色过程
  8. 将切片在蒸馏水中水洗2次,每次3分钟。
  9. 在附带的12毫升的染色缸中混合6ml双蒸水及溶液-4,溶液-5各2毫升,然后将切片放入混合溶液中。盖紧染色缸,等待10分钟。
  10. 在蒸馏水中冲洗切片2次,每次3分钟。
  11. 将切片在50%,75%和95%酒精中梯度脱水各4分钟,然后在100%酒精中脱水两到三次,每次3-5分钟。
  12. 将切片在二甲苯中透明两次,每次4-5分钟,然后使用盖玻片及高浓度二甲苯中性树脂封片剂封片,待干燥后即可在显微镜下观察。
    V. 使用石蜡切片机的替代解决方案
  13. 深度麻醉动物。不要使用缓冲液或固定液灌注动物。小心快速取出组织(脑,脊髓)。大型标本应用锋利的刀片切成厚度约10毫米的组织块。用双蒸水冲洗组织2-3秒,以除去表面的血液。
  14. 将组织转移到至少5倍体积的制备好的浸泡液中,并在室温下避光贮存。
  15. 第二天(12-24小时)更换浸泡液,室温下(20 – 25°C)避光浸泡两周。为了避免非特异性染色,不应延长浸泡时间。
  16. 两周后将组织转移到至少5倍体积的溶液-3中,避光贮存于4℃,12小时后更换溶液-3,在4℃下继续避光贮存24〜72小时。
  17. 在使用溶液-3的处理结束后,将组织转移到一个新的干净容器中,加入双蒸水并在室温下过夜。
  18. 组织水洗完成后,在室温下使用梯度酒精常规脱水,然后将组织移入氯仿中12-24小时进行透明处理。然后用石蜡包埋。(包埋组织推荐使用低熔点的石蜡及真空烤箱,透明处理步骤中,二甲苯也可以用作透明剂,但是使用氯仿处理的组织较为柔软,因此更容易切出较厚的切片,使用二甲苯作为透明剂可导致切片时的组织断裂)。
  19. 水浴槽加入去离子水并设定温度在45-50℃。将石蜡切片机厚度设置为45-80微米,缓慢小心切片,并移入水浴槽,待其完全展开后使用附带的毛笔贴到涂布明胶的载玻片上。
  20. 在37℃斜立载玻片烘干至少15分钟后,再将切片置于60℃的烘箱中2小时或过夜,此后干燥的切片可以在室温下保存三个月。
  21. 将切片在二甲苯中脱蜡3次,每次5分钟。
  22. 然后在100%,95%,70%和50%酒精中重新水化,每一浓度2次,每次5分钟。
  23. 将切片在蒸馏水中水洗2次,每次3分钟。
  24. 在附带的12毫升的染色缸中混合6ml双蒸水及溶液-4,溶液-5各2毫升,然后将切片放入混合溶液中。盖紧染色缸,等待10分钟。
  25. 在蒸馏水中冲洗切片2次,每次3分钟。
  26. 将切片在50%,75%和95%酒精中梯度脱水各4分钟,然后在100%酒精中脱水两到三次,每次3-5分钟。
  27. 将切片在二甲苯中透明两次,每次4-5分钟,然后使用盖玻片及高浓度二甲苯中性树脂封片剂封片,待干燥后即可在显微镜下观察。
    VI. 使用多聚甲醛或甲醛固定组织进行高尔基染色处理的解决方案

如果使用多聚甲醛或甲醛固定的组织进行高尔基染色处理,需要将组织放在流动的自来水中24小时,然后在蒸馏水中再浸泡至少24小时。之后即可按照标准操作步骤开始浸泡处理。

染色结果证明使用多聚甲醛或甲醛固定的组织进行高尔基染色处理, 神经元树突分支和突触棘染色效果是令人满意的。但同时大量神经胶质细胞也出现阳性染色。

虽然我们不建议使用固定的组织进行高尔基染色处理,但在回溯性研究中仅有固定保存的病理标本的情况下,我们的染色试剂盒提供的替代解决方案,将会有其独特的意义。