脂肪染色法
土豆脂肪染色实验报告(3篇)
第1篇一、实验目的通过本次实验，旨在探究土豆中脂肪的分布情况，学习使用苏丹Ⅲ染色法对脂肪进行定性检测，并观察土豆组织中的脂肪颗粒，加深对植物细胞中脂肪积累现象的理解。
二、实验材料与试剂1. 实验材料：- 新鲜土豆- 无水乙醇- 苏丹Ⅲ染液- 70%乙醇- 50%乙醇- 生理盐水- 剪刀、镊子、解剖针、载玻片、盖玻片、显微镜等2. 实验试剂：- 苏丹Ⅲ染液：配制浓度为0.5%的苏丹Ⅲ染液。
- 70%乙醇：用于脱色处理。
- 50%乙醇：用于清洗载玻片。
三、实验方法1. 取新鲜土豆，用剪刀切去两端，沿切面用解剖针挑出小块土豆组织。
2. 将挑出的土豆组织放入载玻片中央，用解剖针轻轻压扁。
3. 向载玻片上滴加苏丹Ⅲ染液，覆盖整个土豆组织，染色时间为5分钟。
4. 取出载玻片，用50%乙醇清洗载玻片，去除多余的染液。
5. 观察染色后的土豆组织，用显微镜观察脂肪颗粒。
6. 将显微镜下观察到的结果记录在实验报告中。
四、实验结果在显微镜下观察，发现土豆组织中的脂肪颗粒呈红色，分布不均匀。
部分细胞内脂肪颗粒较大，部分细胞内则没有明显脂肪颗粒。
五、实验分析1. 土豆作为一种常见的食用作物，其脂肪含量相对较低。
通过苏丹Ⅲ染色法观察，可以清晰地看到土豆组织中的脂肪颗粒。
2. 实验结果表明，土豆组织中的脂肪颗粒主要分布在细胞内，且分布不均匀。
这可能是因为土豆在生长过程中，脂肪的积累与植物的光合作用、呼吸作用等生理过程有关。
3. 在观察过程中，部分细胞内脂肪颗粒较大，这可能是因为这些细胞在生长过程中积累了较多的脂肪。
六、实验讨论1. 本实验使用苏丹Ⅲ染色法对土豆组织中的脂肪进行检测，结果表明该染色法适用于植物组织中脂肪的定性检测。
2. 土豆作为一种低脂食品，其脂肪含量相对较低，但仍然存在一定的脂肪积累。
在日常生活中，适量食用土豆有助于保持身体健康。
3. 本实验结果表明，土豆组织中的脂肪颗粒分布不均匀，这与植物的生长发育过程有关。
显示脂肪的染色方法及其应用
诊断局部感染,收入外科治疗。
使用抗生素治疗1周,右臀部肿胀减轻,右大腿肿胀明显,10日后再行血常规检测,白细胞38.8×109/L,血红蛋白95g/L,血小板30×109/L,淋巴细胞0.03,单核细胞0.10,中性粒细胞0.87,未作涂片镜检,故无幼稚细胞报告,而临床考虑为血液病,做骨髓穿刺,诊断为急性早幼粒细胞性白血病。
临床检验人员应熟悉急性白血病的各种临床表现特点,当血红蛋白、血小板减少,白细胞超越正常参考值,或直方图异常的血标本均应涂片镜检,防止各类白血病细胞漏检。
1.2 人员素质与高科技之间的差距 目前,部分临床检验工作者,对临床使用的进口血液分析仪性能不能全面深入了解和熟练掌握。
如我院最新引进的迈瑞BC-3000plus型全自动血液分析仪在1m in内可测出19项参数,可做出标本含义报告,该机采用国内普遍使用的电阻抗分析原理,依据血细胞经溶血剂作用后,由核和胞质颗粒结构大小决定白细胞分群结果。
由于部分检验人员对仪器性能缺乏足够的认识,误认为仪器白细胞分群结果可代替镜检分类,致白血病细胞漏检情况时有发生[3]。
如1例36岁女性患者,因鼻、牙龈出血来我院就诊,经2次血常规检查,血红蛋白110g/L,血小板32×109/L,白细胞11×109/L,淋巴细胞0.68,单核细胞0.22,中性粒细胞0.10,未作幼稚细胞报告。
医师初诊原发性血小板减少性紫癜,入院治疗第7天骨穿诊断为急性淋巴细胞性白血病,此时外周血幼稚细胞达0.36。
1.3 缺乏高度的责任心 各类白血病的诊断主要依据实验室血液和骨髓象的分析,为得出准确的结果,要求检验人员必须具有高度的责任心,熟练掌握仪器性能、操作规程,检查血液分析结果时才能做到全面、细心,及时发现白血病细胞。
目前,由于受检验人员编制的限制,各大医院的血常规检测工作量较大,如果责任心不强,仅仅是机械的操作,或仅满足简单数字分析,则极易引起白血病漏诊、误诊。
秀丽隐杆线虫脂肪染色的方法比较
秀丽隐杆线虫脂肪染色的方法比较冯婉娟;黄文明;许想平;吴政星【摘要】Obesity and its related diseases are affecting an increasing number of people.Many key fat regulatory genes and fat metabolism pathways found in mammals are conserved inC.elegans.Therefore,C.elegans is a powerful genetic model for fat biology research.Different methods were used to quantitate the fat stores,and the results turn out different.According to the test in fixed worms during fasting or gene mutation,the dye such as Oil Red O,Sudan Black B,Nile Red and C1-BODIPY-C12 could quantitate fat stores correctly.%肥胖及其相关的疾病影响了越来越多的人.在哺乳动物中调节脂肪代谢的因子和脂肪代谢途径在线虫中也是保守存在的,因此线虫是脂肪研究的良好动物模型.在研究线虫脂肪代谢中,存在多种脂肪染色方法,而不同方法所表征的脂肪含量存在一定的差异,通过各种染色方法检测基因突变或者饥饿引起的脂肪含量变化.实验结果表明,采用油红、苏丹黑、尼罗红染料的固定染色法均能够正确地表征线虫的贮存脂肪.【期刊名称】《生命科学研究》【年(卷),期】2012(016)001【总页数】6页(P9-14)【关键词】线虫;脂肪染色;荧光染料;油红;苏丹黑【作者】冯婉娟;黄文明;许想平;吴政星【作者单位】华中科技大学生命科学与技术学院中国湖北武汉430074;华中科技大学生命科学与技术学院中国湖北武汉430074;华中科技大学生命科学与技术学院中国湖北武汉430074;华中科技大学生命科学与技术学院中国湖北武汉430074【正文语种】中文【中图分类】Q504Fat metabolic pathways are conserved between C.elegans and mammals.These pathways include fatty acid synthesis,fatty acid elongation and desaturation,mitochondrial and peroxisomal β-oxidation of fatty acids,and amino acid metabolism[1～5].In addition,C.elegans store fat in droplets in their intestinal cells and hypodermal cells[4],and these fat stores can be directly visualised by microscopy in intact animals due to the transparent bodies of C.elegans.Thus,C.elegans is a powerful system for analyzing the mechanisms of fat storage.There are several methods for examining fat storage and metabolism in C.elegans.An early method for visualising fat storage is using a classic lipophilic dye,Sudan Black B,to stain fixed animals[6,7].In using this stain,lipid droplets become visiible in intestinal and hypodermal tissues.Oil Red O is also a classic lipophilic dye that stains lipid droplets red[8].Aside from colourimetric dyes,another way to stain fat is through the use of lipophilic fluorescent dyes.For example,Nile Red and C1-BODIPY-C12 both fluoresce when in hydrophobicenvironments and thus are used to stain intracellular lipid droplets and can be used to examine fat content in intactliving animals[9,10].When fed C1BODIPY-C12,worms show fluorescence not only fat stores but also other organelles containing lipids.Though these mentioned dyes label fat storage compartments,there is controversy surrounding them,mainly that their results are inconsistent[9,11].An alternative dye-labelling assay for quantifying fat stores is coherent anti-Stokes Raman scattering(CARS)microscopy[12,13],which is a label-free chemicalimagingtechniquethatrelieson intrinsic molecular vibration as a contrast mechanism.Both hypodermal and intestinal fat stores are visualized using this technique.However,the disadvantage of CARS microscopy is that specialised equipment is required and that the assay is very expensive.In this paper,we confirmed that Nile Red and C1-BODIPY-C12 could stain fat storage as efficiently as Oil Red O in fixed worms.Then,we used fixed staining with different dyes to label fat stores in well-fed and fastingC.elegans,where fat stores are present orconsumed,respectively.Finally,using different dyes we tested several mutants which are insulin pathway related or a nuclear hormone receptor of stly,we compared the use of different dyes in fat storage quantification to facilitate researchers who could benefit from such analysis.Nematode strains were obtained from the C.elegans geneticcentre(CGC)unless otherwise stated.All strains were maintained at20℃us ing standard methods[14].The strains used in this study were as follows:wild-type(N2),daf-2(e1370),nhr-49(gk405)and ZXW3 hkdEx3[sur-5::atgl-1::gfp；Rol-6].Oil Red O staining was performed as previously described[9].To permeabilize the cuticle,worms were suspended and washed twice with PBS(phosphate buffered saline)and then suspended in 120 μL ofPBS.Next,an equal volume of 2× MRWB(Modified Ruvkun’s witches brew)buffer containing 2%paraformaldehyde was added,and animals in suspension were rocked for an hour(composition:160 mmol/L KCl,40 mmol/L NaCl,14 mmol/L Na2EGTA,1 mmol/L spermidine HCl,0.4 mmol/L spermine,30 mmol/L Na PIPES at pH 7.4,0.2%βmercaptoethanol).After permeabilizing,worms were re-suspended and dehydrated in60%isopropanol for 15 min at room temperature.After allowing worms to settle,isopropanol was removed,and approximately 1 mL of 60%Oil Red O solution(Cat.No.09755,Sigma-Aldrich,St.Louis,MO,USA)was added to each sample.Samples were incubated overnight while rocking.Oil Red O was prepared as follows:0.5 g of Oil Red O powder was dissolved in 100 mL of isopropanol and equilibrated for several days.Animals were mounted and imaged using an Olympus microscope outfitted with DIC optics.For Sudan Black staining,young adult animals were fixed in2%paraformaldehyde in M9 buffer(3 g KH2PO4,6 g Na2HPO4,5 g NaCl,1 mL 1 mol/L MgSO4,adding H2O to 1 L)with rocking for an hour.Fixed worms were then washed with M9 and dehydrated through an ethanol series (25%,50%,and 70%ethanol).Staining was performed overnight in a 50%saturated solution of Sudan black B in 70%ethanol[15].Stained animals were visualised with an Olympus microscope outfitted with DIC optics.Approximately 500～1 000 nematodes were suspended in 1 mL of water,and 50 μL of freshlyprepared 10% paraformal dehyde solution was then added.Animals and paraformaldehyde solutions were mixed and rocked for an hour.Afterward,rocking was stopped,and worm solutions were allowed to settle.Then,1 mL of 100 μg/L Nile Red in M9 was added to the worm pellet,and the entire solution was incubated for 6 h at room temperature with occasional gentle agitation.Worms were allowed to settle again and then were washed once with M9 buffer.After most of the staining solution had been removed,the fixed worms were mounted onto 2%agarose pads for microscopic observation and photography[15].A stock solution (2 g/L)was made by dissolving C1-BODIPY-C12 in DMSO (Dimethyl sulfoxide).The stock solution was then diluted in 1X PBS to a final concentration of 100 μg/L C1-BODIPYC12,and fixed worms were incubated 6 h in the working solution.After most of the staining solution had been removed,the fixed worms were mounted onto 2%agarose pads for microscopic observation and photography[16].Fixe d Nile Red and C1-BODIPY-C12 stained worms were visualised using an Olympus IX 71 inverted microscope (Olympus,Japan)using a 40x objective(Olympus UPlanSApo series).Fourteen bit images were taken with a CCD camera(Andor DV885).Nile Red yellow (referred to as Nile Red 580)was visualised with 586/20 nm emission filters with excitation light of 491 nm.Image analysis was performed using Image J software(Wayne Rasband,USA).A recent study claimed that Nile Red and C1-BODIPY-C12 did not show fatstorage staining[12].When C.elegans are fed Nile Red,the dye accumulates in lysosome-related organelles[17].Therefore,it was concluded that fixed Nile Red is a better proxy for fat storage visualisation than fed Nile Red.To confirm that fixed C1-BODIPY-C12 and fixed Nile Red are both good indicators of fat stores,we co-localised fixed Nile Red and fixed C1-BODIPY-C12 images.As shown in Fig.1a,we found that fixed Nile Red and fixed C1-BODIPY-C12 co-labelled a population of structures in gut epithelial organelles.Additionally,fixed Nile Red co-localised with Oil Red O and ATGL-1::GFP (Fig.1b and 1c).ATGL-1 encodes a homology of mammalian adipose triglyceride lipase,which is a lipid dropletmarker[18].So fixed Oil Red O staining is a reliable method to measure fat stores.Thus fixed Nile Red and fixed C1-BODIPY-C12 are sufficient dyes to visualise fat stores properly in C.elegans models.We also conducted similar assays which fed Nile Red and ATGL-1::GFP were assessed for co-localisation.According to Fig.1d,fed Nile Red was not co-localised with ATGL-1::GFP,it suggested a poor indicator of fat stores did fed Nile Red act. Mammals consume fat stores to fulfil their energy requirements upon starvation.We used different dyes to examine fat storage changes between well-fed and starved worms.After 12 h of starvation,worms were fixed and stained using Sudan Black B,Oil Red O,Nile Red and C1-BODIPYC12,respectively.In Oil Red O stained worms,well-fed animals had notable staining,while starved animals exhibited reduced staining(Fig.2).Sudan Black-stained worms show similar results(Fig.2).As for fixed Nile Red worms,the fluorescence intensity was brighter in well-fed wormsthan in 12 h starved worms (Fig.2).The situation was the same in fixed C1-BODIPY-C12-stained worms (Fig.2).Taken together,fasting caused fat storage consumption in C.elegans just as in mammals,and different dye-labelled assays showed the reflected fat store consumption in starved worms compared to well-fed worms.The insulin pathway is an important signal pathway to control lipid metabolism.The gene daf-2 encodes an insulin-receptor.Mutation of this gene results in a temperature-sensitive and constitutive dauer(a diapause stage of nematode worms whereby the larva can survive harsh conditions)formation.When daf-2 mutants are grown at permissive temperatures,the adults exhibit increased lifespan and enhanced fat storage[15].To test if the different fat staining assays can reflect the role of DAF-2 in neutral fat mass regulation,we examined daf-2(e1370)mutants using different dye-labelled assays.According to Fig.3,fat content stained by Oil Red O and Sudan Black B in daf-2 loss-of-function mutants were much higher than that of the wild type.Fluorescence dye assays with Nile Red and C1-BODIPY-C12 were performed and fluorescence intensities of fat stores were then quantitated.Results in Fig.4 showed that fluorescence intensity increased in daf-2 mutants compared to that of wild-type worms in both fluorescence dye assays.This increase suggested that the lack of functional daf-2 caused accumulation of fat stores as previously described[15].Taken together,these results suggest that different assays can represent fat masses in C.elegans.The gene nhr-49 encodes a nuclear hormone receptor (NHR),and a recent study indicated that nhr-49 acts as akey regulator of fat usage by modulating fat consumption and fatty acid composition in C.elegans[3,19,20].However,this conclusion rested on the Nile Red live fed staining and was later proven to be a poor indicator of fat content.Therefore,we tested the fat stores of nhr-49 mutants in several assays.Our data showed that Oil Red O and Sudan Black-labelled nhr-49 mutants exhibited normal fat stores as in wild-type animals.We also labelled wild-type and nhr-49 mutants with Nile Red and C1-BODIPY-C12.Results were similar to Oil Red O and Sudan Black staining.This similarity might be due to the fact that NHR-49 protein regulates fatty acid metabolism and only negligibly affects fat stores.Different assays using dye labels could be used to monitor fat storage changes caused by fasting or gene mutation[17].In this paper,we compared fat stores in well-fed and starved worms and specific genetic mutants.As anticipated,fat stores were consumed when wormsfasted.Additionally,all dye-labelled assays used in this study showed increases in fat stores in daf-2 mutants.It is universally acknowledged that daf-2 mutants show this phenotype.For nhr-49 mutants,some research has suggested that nhr-49 animals display abnormally high fatcontent[3,19].However,this conclusion depends on the Nile Red staining in which the dye was fed to animals,which has been proven to be a poor indicator of fat content[17].We tested the fat stores of nhr-49 mutants in several assays and concluded that nhr-49(gk405)mutants exhibit normal abilities to store fat.We summarised different assays of fat labelling with dyes in Table 1.Generally,Oil Red O,Sudan Black B,fixed Nile Red,and fixed C1-BODIPY-C12 are successful in accurately representing fat stores.However,classical fixed Sudan black dye has been shown easily to unsuccessfully label fat stores in C.elegans due to the ethanol-based wash-ing steps during the fixing procedure[15].Oil Red O was supported as an appropriate method to stain and quantify the main fat stores in C.elegans[9].The limitation of this method is that fixation can be variable and difficult to quantify.Certain studies in-dicated that fluorescent dyes,such as Nile Red and C1-BODIPY-C12,could be used to examine fat content in intact living animals.The advantage of Nile Red is the ability to use it in high-throughput screens designed to identify gene inactivations as-sociated with fat reduction or accumulation.Howev-er,more recent research has suggested that fluores-cent dyes fed to worms could not represent neutral lipid content in live worms[12].Therefore,we chose C1-BODIPY-C12 and Nile Red to stain fat stores when used in fixative assays.Both fixed Nile Red and fixed C1-BODIPY-C12 staining performed with short incubation times led to very efficient staining of lipid droplets.Additionally,C1-BODIPY-C12 is more specific than Nile Red staining of lipid droplets,and this method may be suitable for screening assays.Acknowledgement:We thank Caenorhabditis Genetic Centre for wild-type,nhr-49(gk405),and daf-2(e1370)stains and Roy R for sur-5::atgl-1::gfp construct.[1]MCKAY R M,MCKAY J P,AVERY L,et al.C elegans:a model for exploring the genetics of fat storage[J].Developmental Cell,2003,4(1):131-142.[2]WANG J,KIM S K.Global analysis of dauer gene expression in Caenorhabditis elegans[J].Development,2003,130(8):1621-1634.[3]Van GILST M R,HADJIVASSILIOU H,JOLLY A,et al.Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition inC.elegans[J].PLoS Biology,2005,3(2):e53.[4]ASHRAFI K.Obesity and the regulation of fatmetabolism[J].WormBook,2007,1-20.[5]HOLT S J,RIDDLE D L.SAGE surveys C.elegans carbohydrate metabolism:evidence for an anaerobic shift in the long-lived dauerlarva[J].Mechanisms of Ageing and Development,2003,124(7):779-800.[6]RALSER M,BENJAMIN I J.Reductive stress on life span extension inC.elegans[J].BMC Research Notes,2008,(1):19.[7]OGG S,RUVKUN G.The C.elegans PTEN homolog,DAF-18,acts in the insulin receptor-like metabolic signaling pathway[J].MolecularCell,1998,2(6):887-893.[8]SOUKAS A A,KANE E A,CARR C E,et al.Rictor/TORC2 regulates fat metabolism,feeding,growth,and life span in Caenorhabditiselegans[J].Genes&Development,2009,23(4):496-511.[9]O'ROURKE E J,SOUKAS A A,CARR C E,et al.C.elegans major fats are stored in vesicles distinct from lysosome-related organelles[J].Cell Metabolism,2009,10(5):430-435.[10]ASHRAFI K,CHANG F Y,WATTS J L,et al.Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes[J].Nature,2003,421(6920):268-272.[11]WANG M C,O'ROURKE E J,RUVKUN G.Fat metabolism links germline stem cells and longevity in C.elegans[J].Science,2008,322(5903):957-960.[12]YEN K,Le TT,BANSAL A,et al.A comparative study of fat storage quantitation in nematode Caenorhabditis elegans using label and labelfree methods[J].PLoS One,2010,5(9):e12810.[13]KLAPPER M,EHMKE M,PALGUNOW D,et al.Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches[J].Journal of Lipid Research,2011,52(6):1281-1293.[14]BRENNER S.The genetics of Caenorhabditiselegans[J].Genetics,1974,77(1):71-94.[15]KIMURA K D,TISSENBAUM H A,LIU Y,et al.daf-2,an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditiselegans[J].Science,1997,277(5328):942-946.[16]ZHANG S O,BOX A C,XU N,et al.Genetic and dietary regulation of lipid droplet expansion in Caenorhabditis elegans[J].Proceeding of the National Academy of Sciences of the United States of America,2010,107(10):4640-4645.[17]BROOKS K K,LIANG B,WATTS J L.The influence of bacterial diet on fat storage in C.elegans[J].PLoS One,2009,4(10):e7545.[18]NARBONNE P,ROY R.Caenorhabditis elegans dauers need LKB1/AMPK to ration lipid reserves and ensure long-termsurvival[J].Nature,2009,457(7226):210-214.[19]Van GILST M R,HADJIVASSILIOU H,YAMAMOTO K R.A Caenorhabditiselegans nutrient response system partially dependent on nuclear receptor NHR-49[J].Proceeding of the National Academy of Sciences of the United States of America,2005,102(38):13496-13501.[20]HORIKAWA M,SAKAMOTO K.Fatty-acid metabolism is involved in stress-resistance mechanisms of Caenorhabditis elegans[J].Biochemical and Biophysical Research Communications,2009,390(4):1402-1407.【相关文献】[1]MCKAY R M,MCKAY J P,AVERY L,et al.C elegans:a model for exploring the genetics of fat storage[J].Developmental Cell,2003,4(1):131-142.[2]WANG J,KIM S K.Global analysis of dauer gene expression in Caenorhabditiselegans[J].Development,2003,130(8):1621-1634.[3]Van GILST M R,HADJIVASSILIOU H,JOLLY A,et al.Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C.elegans[J].PLoSBiology,2005,3(2):e53.[4]ASHRAFI K.Obesity and the regulation of fat metabolism[J].WormBook,2007,1-20.[5]HOLT S J,RIDDLE D L.SAGE surveys C.elegans carbohydrate metabolism:evidence for an anaerobic shift in the long-lived dauer larva[J].Mechanisms of Ageing and Development,2003,124(7):779-800.[6]RALSER M,BENJAMIN I J.Reductive stress on life span extension in C.elegans[J].BMC Research Notes,2008,(1):19.[7]OGG S,RUVKUN G.The C.elegans PTEN homolog,DAF-18,acts in the insulin receptor-like metabolic signaling pathway[J].Molecular Cell,1998,2(6):887-893.[8]SOUKAS A A,KANE E A,CARR C E,et al.Rictor/TORC2 regulates fatmetabolism,feeding,growth,and life span in Caenorhabditiselegans[J].Genes&Development,2009,23(4):496-511.[9]O'ROURKE E J,SOUKAS A A,CARR C E,et al.C.elegans major fats are stored in vesicles distinct from lysosome-related organelles[J].Cell Metabolism,2009,10(5):430-435.[10]ASHRAFI K,CHANG F Y,WATTS J L,et al.Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory 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第四次实验 脂类化学—苏丹III染色法
在铺片时应掌握好力度，若用力过大，则肠系膜容易被撕破，铺片不成功；若用力过小，则细胞不能被铺呈单层，影响染色及观察。
【实验步骤】
1、断头法处死小鼠，置于解剖盘中，剪开腹腔，用镊子提起小肠，将盖玻片紧贴于肠系膜，用剪刀连同盖玻片和其上粘附的肠系膜剪下，反扣与载玻片上，由盖玻片一边滴入甲醛，使其渗入盖玻片与载玻片间的空隙里，固定20min。
2、吸蒸馏水滴入载玻片与盖玻片之间冲洗，用滴管不断吸去液体，以除去固定液。
3、用70%乙醇溶液代替蒸馏水重复步骤2的操作，再进行一次冲洗。
苏丹染料是偶氮染料，它对脂类的显示是一种简单的物理变化。
苏丹染料是一种脂溶性染料，易溶于乙醇但更易溶于脂肪，所以当含有脂肪的标本与苏丹染料接触时，苏丹染料即脱离乙醇而溶于该含脂肪结构中而使其显色。
脂肪染料一般选用有机溶剂做溶剂，丙酮和乙醇对染料和脂肪都是很好的溶剂，这样可以染色大的脂肪积累块，但是小的脂肪滴会溶解。60%异丙醇当溶剂，可以减轻脂类的溶解。丙二醇或磷酸三乙酯不会溶解脂类物质，但是能溶解染料，是比较理想的溶剂。用这些溶剂配的染料溶液要过滤以去掉沉淀，防止蒸发，因蒸发会引起染料在材料中积累。常用相同溶剂洗掉多余的染料，然后再用水洗，可以防止多余的染料在材料中沉淀。
㈡实验中注意事项有：
1.在抓握小鼠时，应迅速掐住其两耳之间的皮肤，使其头部不可扭动，并用同一只手掐住其背部至尾巴的皮肤，使小鼠整个腹部皮肤紧绷，不能反击。
实验报告 脂类的化学——苏丹三染色
姓名 xxxx 班级 xxxxxx 同组人 xxxxxxxx 科目细胞生物学实验题目脂类的化学——苏丹Ш染色组别第2组一．实验目的1.熟悉脂类的显示技术。
2.了解脂类在细胞中的分布。
二．实验原理脂肪是体内储存能量和供给能量的重要物质，根据其性质可以分为中性脂肪、脂肪酸、胆固醇、鞘磷脂等。
很多细胞都含有脂肪，游离状态的脂肪呈小滴状悬浮于细胞质内，比较显著的如肝细胞。
脂肪小滴可以集合，将细胞质及细胞核挤到一旁，如脂肪细胞。
脂肪不溶于水，易溶于浓乙醇、苯、氯仿和乙醚等，因此制作脂类标本一般不用石蜡切片，而用冰冻切片或者铺片法以保存脂类，固定多用甲醛类固定液。
其染色方法有脂溶性染料显示法、化学显示法和特异染色法等。
脂肪染料一般选用有机溶剂做溶剂，丙酮和乙醇对染料和脂肪都是很好的溶剂，这样可以染色大的脂肪积累块，但是小的脂肪滴会溶解。
60%异丙醇当溶剂，可以减轻脂类的溶解。
丙二醇或磷酸三乙酯不会溶解脂类物质，但是能溶解染料，是比较理想的溶剂。
用这些溶剂配的染料溶液要过滤以去掉沉淀，防止蒸发，因蒸发会引起染料在材料中积累。
常用相同溶剂洗掉多余的染料，然后再用水洗，可以防止多余的染料在材料中沉淀。
用锇酸固定的脂肪不溶于无水乙醇、二甲苯等类似的液体，可用于石蜡切片，但是脂肪的标本一般不用石蜡切片或火棉胶包埋，而用如下方法：冰冻切片，明胶包埋冰冻切片，铺片法。
本实验中使用的脂溶性染料显示法利用苏丹染料中的苏丹III、苏丹IV或者苏丹黑等溶于脂类，而使脂类显色的原理显示脂类，使用时，要注意选择溶剂，要求既要溶解苏丹染料，又不溶掉脂肪。
苏丹染料是偶氮染料，它对脂类的显示是一种简单的物理变化。
苏丹染料是一种脂溶性染料，易溶于乙醇但更易溶于脂肪。
当它与含有脂类的标本接触时，苏丹染料即脱离乙醇而溶于该含脂结构中使其显色。
三．实验仪器及试剂1.仪器解剖盘，解剖剪，镊子，盖玻片，载玻片，显微镜，胶头滴管2.试剂苏丹Ш染液，甲醛钙溶液，70%乙醇溶液，蒸馏水3.材料小白鼠一只四．实验步骤1.断头法处死小鼠，置于解剖盘中。
特殊染色
高碘酸-无色品红法
蒸馏水 偏重亚硫酸钠 170ml 3.96g
活性炭 2.0g(第二天称) 碱性品红(用研钵磨细更佳)加入蒸馏水中溶 解后依次加入盐酸及偏亚硫酸钠，塞住瓶口(锥 形瓶、小口瓶比较好)摇动容器以充分混和(此时 颜色会有明显变化)放置过夜后加入活性炭摇动 数分钟静止1小时后过滤，呈无色或淡稻草黄色 为佳。（不用时冰箱4℃左右保存）
应用：
明确含铁血黄素的存在。如长期的肺郁 血、出血；陈旧性出血灶；肝硬化时慢性脾 郁血的含铁结节；硬化性血管瘤；动脉瘤性 骨囊肿；绒毛色素性滑膜炎等。
胆色素
三氯醋酸三氯化铁（Hall）法：
试剂配制： ㈠Fouchet 液： 甲液：三氯醋酸 25 克 蒸馏水 100 毫升， 乙液：三氯化铁 1克 蒸馏水 10 毫升 两者均宜少量新鲜配制，棕色瓶贮存。 临用时取甲液 30ml 乙液 3ml 等份混合。
Masson 法
•1 切片脱蜡至水.
•2 苏木素染核（可略）
•3 丽春红酸性品红5分钟
•4 快速水洗.蒸馏水洗
•5 1%磷钼酸滴染1-3分钟 •6 倾去余液直接滴加亮绿液5分钟 •7 快速水洗后烤箱烘干,透明封固 •结果：胶原纤维绿色 肌纤维红色
胶原纤维染色的应用 1. 区别胶原纤维与肌纤维。 2. 观察某些病变组织的纤维化及 程度等。
染色方法： 1 常规切片 、脱蜡至蒸馏水 2 0.5%高碘酸氧化5-10分钟 3 流水冲洗数分钟，蒸馏水洗一次 4 无色品红20分钟 5 水洗 5分钟后苏木素染核 6 常规脱水(或烤箱烘干)透明封固 结果：糖原或中性粘液或霉菌等 鲜红色，核蓝色。
注意事项
•1 配制过程中玻璃器皿要干净，试剂要纯。
细胞内脂滴染色方法
细胞内脂滴染色方法细胞内脂滴染色方法是一种常用的技术，用于研究细胞内脂滴的分布、数量和大小。
脂滴是细胞内的脂类储存器，主要由甘油三酯和胆固醇酯组成。
它们在细胞内的分布和代谢与许多生理和病理过程密切相关，如肥胖、糖尿病、脂肪肝等。
因此，准确地检测和定量细胞内脂滴的分布和数量对于理解这些生理和病理过程具有重要意义。
细胞内脂滴染色方法主要有荧光染色和油红染色两种常用技术。
荧光染色是通过使用特定的荧光染料来标记细胞内脂滴。
常用的荧光染料包括BODIPY、Nile Red和Oil Red O等。
这些染料能够与脂滴中的脂类结合，形成荧光染色物。
荧光染色方法的优点是操作简单、快速，且可以通过荧光显微镜直接观察到染色物的分布和数量。
然而，荧光染色的缺点是需要特定的荧光染料和显微镜设备，且染色结果受到许多因素的影响，如荧光染料的浓度、染色时间和细胞处理方法等。
油红染色是一种常用的组织学染色方法，也可以用于细胞内脂滴的染色。
该方法利用油红染料（如Oil Red O）与脂滴中的脂类结合，形成红色染色物。
油红染色方法的优点是操作简单、成本低廉，且染色结果稳定可靠。
然而，油红染色的缺点是不能直接观察染色物的分布和数量，需要通过显微镜观察切片来获取染色结果。
在进行细胞内脂滴染色前，需要对细胞进行预处理。
常用的预处理方法包括固定化、固定化-透明化和固定化-渗透化等。
固定化是将细胞固定在载玻片上，常用的固定剂包括甲醛、乙醛和乙酸等。
固定化-透明化是在固定化的基础上，将细胞透明化，使得荧光染料能够更好地穿透细胞。
固定化-渗透化是在固定化的基础上，通过渗透剂（如Triton X-100）使细胞膜破裂，使荧光染料能够更好地进入细胞内。
选择适当的预处理方法可以提高染色效果和信号强度。
细胞内脂滴染色方法的具体操作步骤如下：1. 处理细胞：根据实验需要，选择合适的细胞系和培养条件。
将细胞培养至适当的生长状态，如对照组和实验组。
2. 固定化：将细胞固定在载玻片上。
脂肪染色
小鼠肠系膜细胞脂肪苏丹Ⅲ染色实验目的：取小鼠肠系膜用脂类染料苏丹Ⅲ染色，观察肠系膜血管周围脂肪细胞的形态和分布，理解苏丹Ⅲ脂类染色的基本原理，熟悉脂类染色的操作方法。
实验原理：脂类细胞化学的主要目的是研究细胞中脂类物质的成分变化以及分布。
在动物细胞中，脂肪是动物体主要的储能物质，很多种细胞都含有脂肪。
通常，细胞中的脂肪和类脂体混合物以游离的液滴状态悬浮在细胞质中，比如肝细胞。
在脂肪含量很高的脂肪细胞中，游离的脂肪液滴可以聚集在一起，占据大部分细胞质空间，将细胞质、细胞核挤到细胞边缘在小鼠肠系膜毛细血管和淋巴管周围，常有单层白色脂肪细胞（对应棕色脂肪细胞）存在这些脂肪细胞的作用主要是以甘油三酸酯和胆固醇酯的形式储存毛细血管从小肠中吸收的部分脂质，待机体需要时再将贮存的脂肪释放到血液中，在特定组织降解并氧化供能。
褐色脂肪细胞由于本身含有大量线粒体，可在脂肪细胞内氧化脂类供能。
细胞中的脂肪不溶于水，易溶于乙醇、氯仿、乙醚等有机溶剂，因此，对脂肪细胞的固定、染色不能使用脂溶剂。
脂肪细胞的固定常使用甲醛类固定剂如甲醛钙，染色使用脂溶性染料如苏丹Ⅲ、苏丹Ⅳ和苏丹黑。
理想脂溶性染料的溶剂应该仅能溶解染料，不溶解脂肪。
脂类染色最常用的染料是苏丹系列染料，本实验即使用苏丹Ⅲ为脂肪显色。
苏丹Ⅲ是一种橙红色偶氮染料，由于其在脂肪中的溶解度高于在乙醇中的溶解度，当用70%乙醇溶解的苏丹Ⅲ饱和溶液浸染脂肪细胞时，苏丹Ⅲ会从70%乙醇中脱离，溶解并集中在脂肪液滴中，使脂肪细胞着色（橘黄色）。
以70%乙醇作为苏丹Ⅲ的溶剂可以减少乙醇对脂肪细胞中脂肪液滴的溶解，染色较大的脂肪块。
染色的主要过程不涉及化学变化。
材料、试剂：小鼠，苏丹Ⅲ70%乙醇饱和溶液（室温），70%乙醇，甲醛钙固定液。
实验方法：将完整展开的小鼠肠系膜连同小肠平铺在载玻片上，以盖玻片盖住肠系膜，剪去多余肠管后，滴加足量甲醛钙固定液固定20min用蒸馏水冲洗玻片肠系膜，除去固定液。
脂类染色
脂类染色脂肪和类脂（磷脂、糖脂、固醇脂等）统称为脂类。
它是构成人体组织的正常成分，不溶于水而易溶于酒精、乙醚、氯仿等脂溶剂中。
在化学组成上，脂类属于脂肪酸的酯或与这些酯有关的物质。
脂类的主要功能是氧化供能。
脂肪主要存积于脂肪组织中，并以油滴状的微粒存在脂肪细胞浆内。
在病理检验中，脂类染色法最常用以证明脂肪变性，脂肪栓子以及肿瘤的鉴别。
脂类染色使用最广泛的染料是苏丹染料，最常用的有苏丹Ⅲ，苏丹Ⅳ，苏丹黑及油红O等。
脂肪被染色，实际上是苏丹染料被脂肪溶解吸附而呈现染料的颜色。
经研究认为组织中脂质在液态或半液态时，对苏丹染料着色效果最好。
根据这一原理，适当提高温度（37℃-60℃）对组织切片染色效果是有好处的。
脂类染色，用冰冻或石蜡切片，以水溶性封固剂封固，如甘油明胶和阿拉伯糖胶等。
一、苏丹Ⅲ(Sudan Ⅲ)染色法：操作方法：（1）固定于10%甲醛的组织。
（2）水洗后采用冰冻或石蜡切片。
（3）经蒸馏水后，浸染于Harris苏木素或明矾苏木素中淡染1-2分钟。
（4）自来水冲洗。
（5）水洗后，移入70%酒精5秒钟。
（6）投入苏丹Ⅲ染液中约30分钟或更长时间，置于56℃温箱中。
（7）在70%酒精中洗涤5-10秒钟。
（8）洗于蒸馏水中，然后将切片移于载玻片上。
（9）切片移于玻片上，将切片周围的水分小心擦掉。
（10）甘油明胶封固。
结果：脂肪呈橙红色或鲜红色或黑色，胆脂素呈淡红色，脂肪酸不着色，细胞核呈蓝色。
试剂配制：1. 苏丹Ⅲ染液配法：将0.15克苏丹Ⅲ溶解于100ml70%酒精或纯丙酮和70%酒精混合液中（各50ml），临用时过滤，所得滤液即为饱和浓度。
注：浸染时，容器必须盖好，否则酒精或丙酮挥发，染料沉淀。
2.甘油明胶配制：明胶 40g蒸馏水 210ml甘油 250ml石碳酸结晶 5ml先将明胶浸入蒸馏水中2小时或更长时间，然后加甘油和石碳酸，加热15分钟，摇搅直至混合液均匀为止。
二、苏丹Ⅳ(Sudan Ⅳ)染色法：苏丹Ⅳ又名猩红，是苏丹Ⅲ的衍生物，作为脂肪染剂，经各地实验证明，其结果优于苏丹Ⅲ。
必修1实验“脂肪的检测”方法改良
必修1实验“脂肪的检测”方法改良【摘要】：“检测生物组织中的脂肪”是高中生物必修1中的实验之一，在多年的实验教学过程中,我们感到教材书中所介绍的实验方法,无论从实验内容还是从实验的具体操作过程来说,都显得比较抽象,而且难以操作。
本实验特针对这些问题加以改良。
【关键词】：脂肪检测，显色反应在人教版《生物. 必修1》第二章第一节，安排了一个鉴定实验---根据显色反应的原理来检测生物组织中的糖类、脂肪和蛋白质，其中关于脂肪的检测有两种方法，方法一如下：向待测组织样液中滴加3滴苏丹Ⅲ染液，观察样液被染色的情况。
该方法只介绍了以花生种子匀浆作为实验材料，用苏丹Ⅲ染色的操作方法和结果，学生往往对于苏丹Ⅲ和苏丹Ⅳ染色的不同现象难以形象的认识，加之此方法需要比较多的花生匀浆材料，且清洗麻烦，现象不明显，在学生分组实验中效果不理想。
在教学过程中，我根据自己的经验对此实验进行了改良，使实验效果非常明显，且操作简单。
具体描述如下：一．实验材料器具直径为15cm的滤纸、花生匀浆、花生油、苏丹Ⅲ、苏丹Ⅳ、滴管二．方法步骤1.将滤纸从中间一分为二。
这样便于节省滤纸。
2.在半圆形滤纸上，从左到右分别各滴加1-2滴花生匀浆和花生油。
3.分别在花生匀浆和花生油滴上滴加苏丹Ⅲ、苏丹Ⅳ进行染色，并在旁边的空白位置各滴加1滴苏丹Ⅲ、苏丹Ⅳ染液作为对照。
4.1分钟后观察实验现象。
三．实验结果可以明显的看出，无论是花生匀浆还是花生油用苏丹Ⅲ染色后都是呈现出橘黄色，用苏丹Ⅳ染色后都呈现出红色，而用于对照的苏丹Ⅲ、苏丹Ⅳ在滤纸上呈粉色。
颜色非常明显，易于区分。
如下图所示：四．实验改良之处本实验相对于教材《必修1－分子和细胞》（人教版）中的实验而言，具有创新性和实用性，就算是条件最简陋的生物实验室都可以完成，具体可从以下几个方面分析：1.选用滤纸作为实验材料，可以免去清洗实验用具的麻烦，减轻了实验员的工作负担。
将滤纸一分为二，可以避免实验材料的浪费，实际中甚至可以将滤纸一分为四，这样进一步节省了材料。
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脂肪染色
【试剂配制】
苏丹III染液：
苏丹III 0.15g
70%酒精100ml。
两者充分混合后，使苏丹III充分溶解，最后行成饱和溶液备用。
试剂瓶密封，使用时过滤。
【染色方法】
（1）冰冻切片厚度8～15μm；
（2）Harris苏木素染约1分钟；
（3）自来水洗后，用0.5%盐酸乙醇分化，再水洗直至胞核返蓝为止；
（4）蒸馏水洗后移入70%乙醇内浸洗一下；
（6）浸入苏丹III染液中约30分钟或更长时间。
如果置于56℃温箱中可适当缩短时间；
（7）在70%乙醇分化数秒钟；
（8）待切片在空气中稍凉干或用冷风机吹干；
（9）及时用明胶甘油封片。
【结果】脂肪呈橘红色，脂肪酸不着色，胞核淡蓝色。