帮忙修正翻译~~

anxiaobao

All mice were deeply anesthetized with isoflurane and
intracardially perfused with physiological saline followed
with ice-cold phosphate-buffered 4% paraformaldehyde
(pH 7.4). The lumbar 4th and 5th spinal cord was dissected
and postfixed in the same fixative for 4 hours at 4°C.
Samples were then cryoprotected in 30% sucrose for 24
hours at 4°C. The sections were cut with a sliding microtome
at a thickness of 40
m. Immunohistochemical
staining for calcitonin gene-related peptide (CGRP), phosphorylated
extracellullar signal-regulated kinases (p-ERK),
and phosphorylated Ca2
/calmodulin-dependent protein
kinase type 2 (p-CaMK-II) were performed with an Elite
ABC Kit (Vector Laboratories, Burlingame, California).
Sections were first rinsed with 0.1 M phosphate-buffered
saline (PBS) 3 times for 10 minutes each, then preincubated
in 0.1 M PBS containing 1% bovine serum albumin (BSA)
and 0.2% Triton X-100 for 30 minutes. After rinsing twice
with 0.1 M PBS containing 0.5% BSA for 10 to 15 minutes
each, sections were incubated with antibodies against
CGRP, p-ERK, and p-CaMK-II diluted with 0.1 M PBS
containing 0.5% BSA and 0.05% sodium azide at 4°C. After
overnight incubation, the sections were rinsed and incubated
with biotinylated secondary antibody diluted 1:200
with 0.1 M PBS containing 0.5% BSA for 1 hour at room
temperature. After rinsing, the sections were incubated
with ABC reagent diluted 1:50 with PBS for 1 hour at room
temperature and then rinsed with PBS followed with 0.1 M
phosphate buffer. Finally, the sections were incubated in
SIGMA FAST DAB kit (Sigma Chemical Co., St. Louis,
Missour) until the desired stain intensity developed. The
sections were rinsed with 0.1 M PB and then mounted on
gelatin-coated slides, and dehydrated with alcohol and
xylene. Antibody expression pattern was calculated and
analyzed by the observer, who was blinded to the study
using Image Pro 6.2 software (Media Cybernetics, Inc.,
Bethesda, Maryland).
Western Blot Analysis
After dissecting the L4 to 5 spinal cord, the tissues were
washed twice in cold PBS and homogenized with a sonicator
in lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA) supplemented with protease
inhibitors. An aliquot (30 or 40
g of protein per lane)
was resolved by 10% or 12% SDS-PAGE and blotted to a
polyvinyl difluoride transfer membrane. Western immunoblotting
was performed with primary antibodies diluted in
5% nonfat dry milk in Tris-buffered saline and then with the
secondary antibodies of hydroxyperoxide-linked antirabbit or
antimouse IgG by using ECL systems (Amersham Biosciences,
Piscataway, New Jersey). Monoclonal antibodies to
p-ERK and p-CaMKII were purchased from Cell Signaling
Technology (Beverly, Massachusetts) and polyclonal antibodies
to -actin were obtained from Santa Cruz Biochemical
(Santa Cruz, California).
求牛人帮忙看下，不做实验，所以这些技术不怎么懂，翻译得比较烂，大家帮忙看看有啥可以改进的地方
以下是本人的翻译：

所有的老鼠使用异氟醚深度麻醉后，心内灌注生理盐水，接下来是冷的4％多聚甲醛磷酸盐缓冲液（pH值7.4）固定。解剖第四和第五腰椎间的脊髓，然后在4摄氏度相同的固定液中固定4小时，然后在30%的蔗糖溶液中去固定24小时。用厚度40微米的滑动切片机切片，用免疫组化的方法测定CGRP,P-ERK,P-CAMKII 机器为EliteABC（Vector Laboratories, Burlingame, California）。

切片用0.1M的PBS溶液漂洗3次，每次10分钟，然后使用0.1M包含BSA的PBS进行30分钟预处理，然后用含0.5%BSA的0.1M PBS漂洗2遍，每次10到15分钟，然后切片用CGRP,P-ERK,P-CAMKII抗体孵育，这些抗体是使用0.1M含0.5BSA和0.05%的钠盐的PBS进行孵育在4°c条件下，孵育过夜后，然后室温下使用含0.5%BSA的0.1M PBS稀释过的二抗进行孵育1小时，漂洗后，切片被用PBS1：50的比例稀释的ABC试剂在室温下孵育一个小时。然后使用PBS漂洗。最后，切片在SIGMA FAST DAB (Sigma Chemical Co., St. Louis,）里孵育，直到染色强度足够。然后用0.1M的PB漂洗，然后固定在有明胶涂层的载玻片上，然后用乙醇和二甲苯脱水，然后对实验双盲的人员对抗体的各种表达模式进行观察统计，使用Image Pro 6.2 software软件进行分析。

anxiaobao

大家发表点意见也行啊。。。。
是不是太长了点，翻译的有点复杂啊
其实就是免疫组化和免疫印迹的步骤。。。
没做过实验，不知道这样翻译是不是够专业。。

anxiaobao

免疫组化
所有老鼠使用异氟醚深度麻醉后，心内灌注生理盐水，接下来用预冷的4％多聚甲醛磷酸盐缓冲液（pH值7.4）灌注固定。解剖取出第四和第五腰椎间的脊髓，在相同的固定液中4℃固定4小时，然后在30%的蔗糖溶液中浸泡24小时。使用滑动切片机切片，切片厚度为40微米， CGRP、p-ERK、p-CaMK-II 的免疫组化染色使用Elite ABC 试剂盒（Vector Laboratories, Burlingame, California）。切片先用0.1M PBS溶液漂洗3次，每次10分钟，然后使用含1%BSA和0.2% Triton X-100的0.1M PBS进行30分钟预孵育，再用含0.5%BSA的0.1M PBS漂洗2遍，每次10到15分钟。然后切片在4℃下用抗CGRP、p-ERK、p-CAMK-II抗体孵育，抗体使用含0.5%BSA和0.05%叠氮钠的0.1M PBS稀释，孵育过夜。隔天将切片漂洗后在室温下用含0.5%BSA的0.1M PBS稀释的生物素标记二抗孵育1小时（1：200稀释），进一步漂洗后，在室温中用被PBS1：50稀释的ABC试剂孵育一小时，然后使用0.1M PBS漂洗。最后，将切片放入SIGMA FAST DAB (Sigma Chemical Co., St. Louis,）试剂中孵育，直至达到所需的染色强度后取出，经0.1M的PBS漂洗后，固定在明胶包被的载玻片上，用乙醇和二甲苯脱水。最后由对实验双盲的人员对抗体的表达格局进行观察统计，并采用Image Pro 6.2 software软件进行分析。

Western Blot 分析
取出L4-5间的脊髓，将组织用冷的PBS缓冲液漂洗2次，放入预先加有蛋白酶抑制剂的裂解液中用超声匀浆机匀浆裂解，蛋白用10%或者12%SDS聚丙烯酰胺凝胶电泳（30-40ug/孔），将电泳后凝胶上的蛋白质转印至PVDF膜上。一抗用含5％脱脂奶粉的Tris缓冲液稀释，二抗为HRP标记的抗鼠或抗兔IgG，检测采用ECL系统(Amersham Biosciences, Piscataway, New Jersey)。p-ERK,p-CAMKII的单抗体购自Cell Signaling Technology (Beverly, Massachusetts)，β-actin多抗购自 Santa Cruz Biochemical(Santa Cruz, California)。




我找做实验的老师帮忙翻译的，大家可借鉴下~~