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).
