Quantitation of row 1 proteins during after tubocurarine block in culture

Each file is for a single mature row 1 complex member, examining its distribution in mouse inner hair cells in the presence or absence of transduction channel block. Cochleas were dissected at P4.5 and cultured in in vitro for two days in control solution or 100 µM tubocurarine. The first two to three tabs in each file represent data from different experiments. Each block of 12 measurements is from a single hair bundle. The next-to-last tab has the average of all row 1 + 2 measurements, giving total expression in those rows. The last tab compares rows 1 and 2.
We estimated the total immunofluorescence signal in the top 1-2 µm of each tip. Airyscan z-stacks were imported into Fiji Software, which was used for all analysis steps. For analysis of P7.5 mutant mice, average Z-projections of Airyscan stacks were made that included row 1 and row 2 tips in the same projection. For analysis of P21.5 mutant mice, due to increased height variability, Regions of Interest (ROIs) were selected at row 1 or row 2 tips within individual x-y slices from the z-stacks. Images were kept as multi-channel stacks and phalloidin was used to guide selection at the tips of each row. Regions of interest (ROIs) used circles that encompassed most of each tip. Area and average intensity measurements were made from ten or more row 1 tips and ten or more row 2 tips, and blank measurements were taken from volumes outside the stereocilia and above the epithelium. The total signal (area times average intensity) in each stereocilia tip volume was calculated after subtracting the background. For each hair bundle, the average signal in rows 1 and 2 was calculated, which was used to normalize the individual row 1 and row 2 stereocilia tip signals within each bundle. The average signal row 1 and 2 signal for each bundle was also used for comparison of expression levels at different developmental times or between control and mutant mice. For comparisons between control and tubocurarine block, the average signal for control bundles on a given experimental day was used to normalize all samples, allowing comparison of controls and channel block with identical acquisition parameters.

本数据集的每个文件对应单个成熟的第1行复合体组分，用以探究其在存在或不存在转导通道阻断(transduction channel block)时，在小鼠内毛细胞中的分布情况。实验于出生后第4.5天（P4.5）分离耳蜗，并在对照溶液或100 μM筒箭毒碱(tubocurarine)溶液中体外培养两天。每个文件的前2至3个工作表代表不同实验获得的数据；每12次测量组成的区块均来自单根毛束(hair bundle)。倒数第二个工作表包含第1行与第2行所有测量结果的平均值，用以表征这两行的总表达量；最后一个工作表则对第1行与第2行的表达量进行比较。
我们对每个静纤毛(stereocilia)顶端1~2 μm区域内的总免疫荧光信号(immunofluorescence signal)进行了定量。将Airyscan z堆叠图像(Airyscan z-stacks)导入Fiji软件(Fiji Software)，以该软件完成所有分析步骤。针对P7.5突变小鼠(mutant mice)的分析，我们对包含第1行与第2行静纤毛顶端的Airyscan堆叠图像进行平均Z投影(Z-projections)处理。针对P21.5突变小鼠的分析，由于静纤毛高度差异增大，我们从z堆叠图像的单张x-y切片中选取第1行或第2行静纤毛顶端作为感兴趣区域(Regions of Interest, ROIs)。图像以多通道堆叠形式保留，采用鬼笔环肽(phalloidin)辅助选取各行静纤毛顶端。所选用的感兴趣区域为圆形，可覆盖大部分静纤毛顶端。我们对10个及以上的第1行静纤毛顶端和10个及以上的第2行静纤毛顶端进行面积与平均强度测量，并在静纤毛外侧及上皮组织(epithelium)上方的区域采集空白对照测量值。在扣除背景(background)信号后，计算每个静纤毛顶端体积内的总信号（面积乘以平均强度）。针对每个毛束，我们计算其第1行与第2行的平均信号，用于对同一毛束内的单个第1行及第2行静纤毛顶端信号进行标准化处理。每个毛束的第1行与第2行平均信号还用于比较不同发育时期或对照组与突变小鼠组之间的表达水平。在对照与筒箭毒碱阻断组的比较中，我们以特定实验日的对照组毛束平均信号对所有样本进行标准化，从而可在相同采集参数下对对照组与转导通道阻断组进行比较。