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标题: 请教大家冰切片苔盼蓝染色方法 [打印本页]

作者: meizhou    时间: 2009-2-14 12:00     标题: 请教大家冰切片苔盼蓝染色方法

我做的是骨骼肌组织冰冻切片,现在打算检测下细胞坏死情况,查找很多文献有用的甲苯胺兰,有的用的苔盼蓝染色,还有的用的NBT染色。
可是这些染色方法我都没做过,百度搜索发现都没有冰切组织的,yizhao老师老师是这个论坛最热心的老师,专业又非常扎实,希望能够帮我看看。也希望做过的老师们进来指点一下,不胜感激 !

[ 本帖最后由 meizhou 于 2009-2-21 12:32 编辑 ]
作者: meizhou    时间: 2009-2-20 21:27

今天做了两次苔盼蓝染色,用1%苔盼蓝染液(溶于1%的硼砂中),染色时间设置为2分钟。结果发现两次都掉片子!以前做过很多染色都没掉过片子(只有ATP酶染色碱性太重时出现过),是不是pH值有问题哦 ?
作者: meizhou    时间: 2009-2-20 21:28

可是这个方法是我从很多文献里看到的啊!文献上说是60度反应1分钟,我试过,掉片子很严重,没想到常温2分钟还是掉。不知道咋办啊?
作者: meizhou    时间: 2009-2-20 21:32

我的试剂是进口分装Amersco的,今天新配的溶液。步骤是1克苔盼蓝和1克硼砂,用研钵磨细,溶解于100毫升水中,过滤。骨胳肌冰冻切片-20度冰箱拿出来,晾片10分钟,染色2分钟,水洗。到这一步就发现掉片了,下面也没脱水和通透。
作者: meizhou    时间: 2009-2-21 12:31

我刚才测了昨天染液的pH值是9.4,偏碱性的。也不是碱性特别重,那怎么会掉片这么严重呢?我从前做ATP酶染色10.6处理5分钟都不掉的呀!
2.3. Histology and histochemistry
Animals were anesthetized with ethyl ether, weighed and
both right and left TAs, gastrocnemius and soleus muscles
were removed. To evaluate a possible systemic skeletal
muscle necrosis, it was also removed two muscles located
distant of the site of the toxin injection: masseter and longissimus
dorsi (sample obtained from lumbar region).
Forward, the muscles were immediately frozen in melting
isopentane and stored in a freezer at 2568C. Frozen muscles
were cut through the middle belly region (10 mm crosssections)
using a cryostat (Microm HE, Germany). About
2 mm of each muscle was evaluated by serial cross-sections.
Afterwards, some longitudinal sections (10 mm) were also
obtained. Histological sections were stained with 1% Toluidine
Blue/1% Borax or incubated for acid phosphatase reaction
(ac-phosphatase, Barka and Anderson, 1962).
Toluidine Blue was used to evaluate the morphological
pattern of the muscles, as previously described (Morini etal., 1998; Salvini et al., 1999; Minamoto et al., 1999) and acphosphatase
to identify signals of tissue necrosis and phagocitosis.
It is well known that normal muscle ®bers do not
have positive ac-phosphatase reaction which indicates high
concentration of lysosomes, which is considered an evident
proof of tissue necrosis and phagocitose (Carpenter and
Karpati, 1984).
It is well know that the skeletal muscles are composed by
different kinds of muscle ®ber types. Red muscles are
composed predominantly by muscle ®bers rich in myoglobin,
mitochondria and blood capillaries, which are also classi
®ed as oxidative ®bers and ®ber types I and IIA. Type I
®bers are slow-twitch ®bers. White muscles are composed
by type IIA, IID/X and IIB ®bers, that are classi®ed as fasttwitch
®bers and have predominantly glycolytic metabolism
(for review see Pette and Staron, 1990). In this study,
muscles composed predominantly by fast-twitch ®bers
(TA, masseter, longissimus dorsi, white gastrocnemius)
and slow-twitch ®bers (soleus and red gastrocnemius)
were evaluated. Gastrocnemius muscle was specially
choose for this study because it has two distinct regions:
the red portion is composed predominantly by oxidative
muscle ®bers (51% type I and 35% type IIA), while the
white portion is exclusively composed by glycolytic ®ber
types IID/X (8%) and 92% type IIB (92%) (see Delp and
Duan, 1996 for the muscle composition).




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